Vimentin Directed Diagnostics And Therapeutics For Multidrug Resistant Neoplastic Disease

ABSTRACT

Disclosed are methods for detecting multidrug resistance in neoplastic or damaged cells or multidrug resistant (MDR) neoplastic or damaged cells by detecting an increase in the cell surface expression of vimentin protein in such cells as compared to the level of cell surface expression of vimentin protein in a normal cell or a non-MDR neoplastic cell.

This Application claims the benefit of priority to U.S. ProvisionalApplication No. 60/433,480, filed Dec. 13, 2002, the specification ofwhich is incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of diagnostics and therapeutics. Inparticular, this invention relates to the detection and treatment ofneoplastic and/or damaged cells and, in addition, to the detection andtreatment of multidrug resistant neoplastic and/or damaged cells.

1. BACKGROUND OF THE INVENTION

Diseases such as cancer or those caused by pathogen-infection are oftentreated with drugs (e.g., chemotherapeutics and antibiotics). In orderto kill the cancer or diseased cells, the drug(s) must enter the cellsand reach an effective dose so as to interfere with essentialbiochemical pathways. However, some cells evade being killed by the drugby developing resistance to it (termed “drug resistance”). Moreover, insome cases, cancer cells (also called tumor cells or neoplastic cells)and damaged cells (e.g., pathogen-infected cells), or the pathogensthemselves, develop resistance to a broad spectrum of drugs, includingdrugs that were not originally used for treatment. This phenomenon istermed “multidrug resistance” (MDR). There are different types of MDR,each associated with a different biological mechanism, and there arespecific biological “markers” for different types of MDR that areclinically useful for detecting and diagnosing each type of MDR.

MDR can involve cancer cells or infectious pathogens such as viruses,bacteria, parasites and other microorganisms that may be present incells or body fluids. The emergence of the MDR phenotype is the majorcause of failure in the treatment of infectious diseases (see Davies J.,Science 264: 375-382, 1994; Poole, K., Cur. Opin. Microbiol. 4:500-5008, 2001). Patient cross-resistance to different anti-microbialand anti-cancer agents, which are structurally and functionallydistinct, can cause serious problems for pathogen-infected patients.Similarly, the development of multidrug resistant cancer cells is theprincipal reason for treatment failure in cancer patients (seeGottesman, M. M., Ann. Rev. Med. 53: 615-627, 2000).

Multidrug resistance is multifactoral. The classic MDR mechanisminvolves alterations in the gene for the highly evolutionarily conservedplasma membrane protein (P-glycoprotein or MDR 1) that activelytransports or pumps drugs out of the cell or microorganism (Volm M. etal., Cancer 71: 3981-3987, 1993); Bradley and Ling, Cancer MetastasisRev. 13: 223-233, 1994). Both human cancer cells and infectiousbacterial pathogens may develop classic MDR via mechanisms involving theoverexpression of P-glycoprotein due to amplification of the geneencoding P-glycoprotein. The overexpression of P-glycoprotein mRNA orprotein in MDR cancer cells or pathogen-infected cells is a biologicalmarker for MDR. Diagnostic tests and therapeutic methods have beendeveloped that make use of the overexpression of P-glycoprotein markerto diagnose and to treat MDR cancer and pathogen infections (Szakacs G.et al., Pathol. Oncol. Res. 4: 251-257, 1998). However, because variousnormal tissues express different amounts of P-glycoprotein, there aresignificant problems with side effects as any therapy that targetsP-glycoprotein on the cell surface of MDR cancer cells would also affectthose normal tissues that also have a relatively high level ofP-glycoprotein expression, such as liver, kidney, stem cells, andblood-brain barrier epithelium.

“Atypical MDR” is a term used to describe MDR cancer cells or pathogenswherein the mechanism of multidrug resistance is unknown, novel, ordifferent from the classic mechanism involving P-glycoprotein. Forexample, human lung tumors are multidrug resistant but do not havealterations in P-glycoprotein (see Cole S. P. et al., Science 258:1650-1654, 1992). Rather, they express another drug transporter (themultidrug resistance associated protein or “MRP1”). A new mechanism ofMDR was recently described that involves lung resistance relatedprotein, which is a marker for this type of atypical MDR (Rome L. H. etal., PCT Pub. No. WO9962547). Other examples of atypical markers for MDRinclude MRP5, which is a novel mammalian efflux pump for nucleosideanalog drugs (see Fridland and Schuetz, PCT Pub. No. WO0058471) andcertain sphingoglycolipids (see U.S. Pat. No. 6,090,565). It should benoted that MDR cells may express more than one MDR marker (bothclassical P-glycoprotein and atypical markers) simultaneously on thesame cell, and that the markers are expressed independently (GrandjeanF, et al. Anticancer Drugs, 12:247-258, 2001). It is therefore possibleto combine treatments directed against more than one cell surface MDRmarker and potentially against more than one mechanism of MDR.

Intermediate filaments are a major component of the cytoskeleton ofhigher eukaryotic cells. Intermediate filaments are composed of a numberof different structurally related proteins and different intermediatefilament protein genes are expressed in different tissues. Studiesinvolving spontaneous and experimentally produced mutations inintermediate filament genes have demonstrated that intermediatefilaments function to enhance the mechanical stability of epidermal andmuscle cells (Evans R. M., BioEssays, 20: 79-86, 1998). Vimentin(gi/4507895) is a homodimeric intracellular protein found in class IIIintermediate filaments in mesenchymal and other nonepithelial cells andin the Z disk of skeletal and cardiac muscle cells. (See, e.g., Evans,R. M. (1998) Bioassays 20:79-86; Herrmann, H., Aebi, U. (2000) Curr.Opin. Cell Biol. 12:79-90, ibid. (1998) Subcell Biochem 31:319-62.).Vimentin is a phosphoprotein whose phosphorylation is enhanced duringcell division. Both the human and murine vimentin genes have beencharacterized (see, e.g., Kryszke et al. (1998) Pathol. Biol. 46:39-45;Paulin, D. (1989) Pathol. Biol. 37:277-82). Purified eukaryotic vimentinhas a molecular mass of 54 kDa, is soluble in its native form andinsoluble when denatured. Vimentin gene regulation appears toparticipate in several steps of viral infections (reviewed in Kryszke etal. (1998) Pathol. Biol. 46:39-45).

Various cytoskeleton modifications are associated with malignant celltransformation and have been used as prognostic factors. In contrast tothe in vivo situation where vimentin expression is characteristic ofcells of mesenchymal origin, vimentin synthesis is characteristic of allproliferating cells in vitro regardless of their embryonal origin, andis switched off upon differentiation of certain precursor cells.Vimentin gene expression is upregulated in some metastatic tumor cells,and is thus a marker for oncogenic progression (see, Kryszke et al.(1998) Pathol. Biol. 46:39-45; Osborn et al. (1989) Curr. Comm. in Mol.Biol., Cold Spring Harbor Press).

Vimentin and other IF proteins have been used in the histologicalclassification of human tumors (for reviews see Ramaekers et al. (1982)Cold Spring Harb. Symp. Quant. Biol. 46: 331-339; Thomas et al. (1999)Clin. Cancer Res. 5:2698-2703). Vimentin in particular has been used asa marker for de-differentiation in several types of tumors.

Tumor markers that co-localize with vimentin and other intermediatefilaments have been described. For example, De Bernard, Marina(WO0127269) describes a novel marker for neuroblastomas called VIP54, aprotein that is closely associated with vimentin and desmin filaments,and the use of this internal marker for the detection and cellularimaging of intermediate filaments (particularly class-III vimentin ordesmin filaments) as a markers for tumor development and/or progression.

Finally, there are several examples that show changes in the expressedlevel and intracellular distribution of vimentin in different types ofhuman solid tumors and solid tumor cell lines, often in association withthe development of multidrug resistance (Conforti G, et al., Br. J.Cancer, 3:505-511, 1995; Moran E. et al., Eur J. Cancer, 33:652-660,1997).

There remains a need in both humans and animals for detecting, treating,preventing, and/or reversing the development of both classical andatypical MDR phenotypes in cancer cells and non-cancerous damaged cells,regardless of how the MDR arises (e.g., naturally occurring ordrug-induced). In addition, the ability to identify and to make use ofreagents that identify multiple drug resistant cells has clinicalpotential for improvements in the treatment, monitoring, diagnosis, andmedical imaging of multidrug resistant cancer and multidrug resistantdamaged cells.

2. SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery that full-lengthvimentin, a normally intracellular protein, is expressed in full lengthon the cell surface of neoplastic cells and damaged cells, and isexpressed more abundantly on the cell surfaces of multidrug resistant(MDR) neoplastic cells and MDR damaged cells. Although lower levels ofvimentin are expressed on the cell surface of drug-sensitive neoplasticcells, in contrast to other cell surface MDR markers (such asP-glycoprotein), vimentin is expressed in only negligible amounts on thecell surface of normal cells of the body. By “negligible amounts” ismeant fewer than 100 molecules of vimentin on the cell surface. Thus,the invention allows the use of binding agents, to which are boundtoxins or other therapeutic or diagnostic agents, that specifically bindto vimentin without detrimental side effects, since the onlynon-vimentin cells that are being killed are drug-sensitive neoplasticcells or damaged cells; normal cells remain unharmed.

In one aspect, the invention provides a method for detecting multidrugresistance or multidrug resistance potential in a test neoplastic cellby measuring a level of cell surface-expressed vimentin protein in thetest neoplastic cell of a given origin or cell type, and comparing it tothe level of cell surface-expressed vimentin in a nonresistantneoplastic cell of the same origin or cell type. If the level of cellsurface-expressed vimentin in the test neoplastic cell is greater thanthe level of cell surface-expressed vimentin in the nonresistantneoplastic cell of the same given origin or cell type, then the testneoplastic cell is multidrug resistant or has multidrug resistancepotential. In certain embodiments, the level of cell surface-expressedvimentin in the test neoplastic cell is measured by isolating acytoplasmic membrane fraction from the cell and measuring the level ofvimentin in the cytoplasmic membrane fraction. In other embodiments, thelevel of cell surface-expressed vimentin in the test neoplastic cell ismeasured by contacting the cell with an anti-vimentin antibody andmeasuring the level of antibody bound to cell surface vimentin. Forexample, the level of antibody bound to cell surface vimentin may bemeasured by immunofluorescence emission or radiolabel.

In certain embodiments of this aspect of the invention, the testneoplastic cell is a promyleocytic leukemia cell, a T lymphoblastoidcell, a breast epithelial cell, or an ovarian cell. In other embodimentsthe test neoplastic cell is a lymphoma cell, a melanoma cell, a sarcomacell, a leukemia cell, a retinoblastoma cell, a hepatoma cell, a myelomacell, a glioma cell, a mesothelioma cell, or a carcinoma cell. In stillother embodiments of the invention, the test neoplastic cell is from atissue such as blood, bone marrow, spleen, lymph node, liver, thymus,kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, orovary. In further embodiments, the nonresistant neoplastic (control)cell is a drug-sensistive neoplastic cell line such as HL60, NB4, CEM,HSB2 Molt4, MCF-7, MDA, SKOV-3, or 2008.

In another aspect, the invention provides a method for detecting amultidrug resistant cell or cells in a patient by administering to thepatient, a vimentin binding agent operably linked to a detectable label.The label is operably linked to the vimentin binding agent, whichspecifically binds to cell surface-expressed vimentin present on themultidrug resistant cell(s) in the patient, and is then detected,thereby locating the presence of the multidrug resistant cell(s) (ifany) in the patient. In certain embodiments, the vimentin binding agentused is an antibody or fragment thereof. In other embodiments, thevimentin binding agent is a vimentin ligand such as modified LDL, NLK1protein, vimentin, desmin, glial fibrillary acidic protein, orperipherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase2A. In particular embodiments, the vimentin binding agent is a naturalligand, a synthetic small molecule, a chemical, a nucleic acid, apeptide, a protein or an antibody. In other embodiments, the detectablelabel is a fluorophore, a chemical dye, a radioactive compound, achemoluminescent compound, a magnetic compound, a paramagnetic compound,a promagnetic compound, an enzyme that yields a colored product, anenzyme that yields a chemoluminescent product, or an enzymes that yieldsa magnetic product.

In certain embodiments of this aspect, the multidrug resistant cell is aneoplastic cell. In particular embodiments, the neoplastic cell is abreast cancer cell, an ovarian cancer cell, a myeloma cancer cell, alymphoma cancer cell, a melanoma cancer cell, a sarcoma cancer cell, aleukemia cancer cell, a retinoblastoma cancer cell, a hepatoma cancercell, a glioma cancer cell, a mesothelioma cancer cell, or a carcinomacancer cell. In some embodiments, the neoplastic cell is a promyleocyticleukemia cell, a T lymphoblastoid cell, a breast epithelial cell, or anovarian cell. In particular embodiments, the patient is a human, such asa human patient that is suffering from a disease or disorder caused bythe presence of the multidrug resistant cell.

In another aspect, the invention provides kits for diagnosing ordetecting multidrug resistance in a test neoplastic cell. The kitsinclude one probe for the detection of vimentin and a second probe forthe detection of another multidrug resistance marker such asnucleophosmin or HSC70. In certain embodiment, these kits of theinvention include a first probe for the detection of vimentin and asecond probe for the detection of another multidrug resistance markersuch as MDR1, MDR3, MRP1, MRP5, or LRP. In particular embodiments, thekits include anti-vimentin antibody as the probe for detecting vimentin.In other embodiments, the kits include a vimentin ligand such as LDL,NLK1 protein, vimentin, desmin, glial fibrillary acidic protein, andperipherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase2A. In further embodiments, the kits include a nucleophosmin antibody oran HSC70 antibody as probes for detecting the second, non-vimentinmultidrug resistance marker. In other embodiments, the second probe maybe a nucleophosmin ligand (such as protein kinase R (PKR), RNA,retinoblastoma protein, IRF-1, or a nuclear localization signals (NLS)such as the N-terminal nuclear localization signal of Rex protein) or anHSC70 ligand (such as Alzheimer's tau protein, BAG-1, smallglutamine-rich tetratricopeptide repeat-containing protein (SGT), (aa642-658) of rotavirus VP5 protein, auxilin, or the immunosuppressant5-deoxyspergualin (DSG)).

In another embodiment of this aspect of the invention, the kit includesa probe that detects vimentin present on the surface of the testneoplastic cell. In other embodiments, the kit includes a second probethat detects another (non-vimentin) marker present of the surface of thetest MDR neoplastic cell. In certain embodiment, the kit includes asecond probe that is an MDR1 antibody, an MDR3 antibody, an MRP1antibody, an MRP3 antibody, or an LRP antibody.

In another aspect, the invention provides a cell surface vimentin insitu detection probe for the detection of cell surface vimentin in apatient. This cell surface vimentin probe has a vimentin bindingcomponent and a detectable label for detection in situ (e.g., aTechnetium label). In some embodiments, the vimentin binding componentis an antibody.

In yet another aspect, the invention provides a cell surfacevimentin-targeted agent for treating or preventing a multi-drugresistant neoplasm. This vimentin-targeted agent includes both avimentin binding component and a therapeutic component which acttogether such that the vimentin binding component targets thetherapeutic component to the multi-drug resistant neoplasm and therebytreats the multi-drug resistant neoplasm. In certain embodiments, thevimentin binding component is an anti-vimentin antibody. In otherembodiments, the vimentin binding component is a vimentin ligand such asLDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein,peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase2A. In particular embodiments, the vimentin binding component is anatural ligands, synthetic small molecules, chemicals, nucleic acids,peptides or protein.

In certain useful embodiments of this aspect of the invention, thetherapeutic component is a chemotherapeutic agent such as Actinomycin,Adriamycin, Altretamine, Asparaginase, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide, Fludarabine,Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide,Imatinib, Irinotecan, Lomustine, Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,Vinblastine, Vincristine, or Vinorelbine. In particular embodiments, thetherapeutic component is in a liposome formulation.

In other embodiments, the therapeutic component is a radioisotope suchas ⁹⁰Y, ¹²⁵I, ¹³¹I, ²¹¹At or ²¹³Bi.

In still other embodiments, the therapeutic component is a toxin capableof killing or inducing the killing of the targeted multi-drug resistantneoplastic cell. Such toxins for use in the invention includePseudomonas exotoxin, diphtheria toxin, plant ricin toxin, plant abrintoxin, plant saporin toxin, plant gelonin toxin and pokeweed antiviralprotein.

In particularly useful embodiments, the vimentin binding component ofthe cell surface vimentin-targeted therapeutic agent binds to thesurface of the target cell, and the therapeutic element is internalizedand arrests growth of the cell, compromises viability of the cell orkills the cell.

In another aspect, the invention provides a method of treating orpreventing a multidrug resistant neoplasm in a subject by administeringany of the cell surface vimentin-targeted therapeutic agents describedabove. In certain embodiments of this aspect, the neoplasm is a breastcancer, an ovarian cancer, a myeloma, a lymphoma, a melanoma, a sarcoma,a leukemia, a retinoblastoma, a hepatoma, a glioma, a mesothelioma, or acarcinoma. In further embodiments, the neoplasm is from a tissue such asblood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain,skin, gastrointestinal tract, eye, breast, prostate, or ovary. Inparticular embodiments, the subject is a human patient, such as a humanpatient suffering from a disease or disorder caused by the presence ofthe multi-drug resistant cell.

In yet another aspect, the invention provides vaccines for treating orpreventing multi-drug resistant neoplasms, comprising a vimentinpolypeptide, or vimentin polypeptide subsequence thereof, and at leastone pharmaceutically acceptable vaccine component. In certainembodiments, the vimentin polypeptide or polypeptide subsequence is ahuman vimentin polypeptide sequence having an amino acid sequence of SEQID NO.: 1. In particular embodiments, the vimentin polypeptidesubsequence is at least eight amino acids long, and, in certainembodiments, functions as a hapten.

In certain embodiments, the vaccine formulation includes an adjuvant orother pharmaceutically acceptable vaccine component. In particularembodiments, the adjuvant is aluminum hydroxide, aluminum phosphate,calcium phosphate, oil emulsion, a bacterial product, whole inactivatedbacteria, an endotoxins, cholesterol, a fatty acid, an aliphatic amine,a paraffinic compound, a vegetable oil, monophosphoryl lipid A, asaponin, or squalene.

In another aspect, the invention provides a method of treating orpreventing a multidrug resistant neoplasm in a subject by administeringany of the vimentin vaccines described above. In certain embodiments ofthis aspect, the neoplasm to be treated is a breast cancer, an ovariancancer, a myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, aretinoblastoma, a hepatoma, a glioma, a mesothelioma, or a carcinoma. Infurther embodiments, the neoplasm is from a tissue such as blood, bonemarrow, spleen, lymph node, liver, thymus, kidney, brain, skin,gastrointestinal tract, eye, breast, prostate, or ovary. In particularembodiments, the subject is a human patient, such as a human patient issuffering from a disease or disorder caused by the presence of themulti-drug resistant cell.

In yet another aspect, the invention provides a method for detectingwhether a test cell is neoplastic by measuring the level of cellsurface-expressed vimentin protein in the test cell of a given origin orcell type, and comparing it to the level of cell surface-expressedvimentin in a normeoplastic cell of the same origin or cell type. If thelevel of cell surface-expressed vimentin in the test cell is greaterthan the level of cell surface-expressed vimentin in the normeoplasticcell of the same given origin or cell type, then the test cell isneoplastic.

In certain embodiments, the level of cell surface-expressed vimentin inthe test cell is measured by isolating a cytoplasmic membrane fractionfrom the cell and measuring the level of vimentin in the cytoplasmicmembrane fraction. In other embodiments, the level of cellsurface-expressed vimentin in the test cell is measured by contactingthe cell with an anti-vimentin antibody and measuring the level ofantibody bound to cell surface vimentin. For example, the level ofantibody bound to cell surface vimentin may be measured byimmunofluorescence emission or radiolabel.

In certain embodiments of this aspect of the invention, the test cell isa promyleocytic leukemia cell, a T lymphoblastoid cell, a breastepithelial cell, or an ovarian cell. In other embodiments the test cellis a lymphoma cell, a melanoma cell, a sarcoma cell, a leukemia cell, aretinoblastoma cell, a hepatoma cell, a myeloma cell, a glioma cell, amesothelioma cell, or a carcinoma cell. In still other embodiments ofthe invention, the test cell is from a tissue such as blood, bonemarrow, spleen, lymph node, liver, thymus, kidney, brain, skin,gastrointestinal tract, eye, breast, prostate, or ovary.

In another aspect, the invention provides a method for detecting aneoplastic cell or cells in a patient by administering to the patient, avimentin binding agent operably linked to a detectable label. The labelis operably linked to the vimentin binding agent, which specificallybinds to cell surface-expressed vimentin present on the neoplasticcell(s) in the patient, and is then detected, thereby locating thepresence of the neoplastic cell(s) (if any) in the patient. In certainembodiments, the vimentin binding agent used is an antibody or fragmentthereof. In other embodiments, the vimentin binding agent is a vimentinligand such as modified LDL, NLK1 protein, vimentin, desmin, glialfibrillary acidic protein, or peripherin, fimbrin, RhoA-binding kinasealpha, or protein phosphatase 2A. In particular embodiments, thevimentin binding agent is a natural ligand, a synthetic small molecule,a chemical, a nucleic acid, a peptide, a protein or an antibody. Inother embodiments, the detectable label is a fluorophore, a chemicaldye, a radioactive compound, a chemoluminescent compound, a magneticcompound, a paramagnetic compound, a promagnetic compound, an enzymethat yields a colored product, an enzyme that yields a chemoluminescentproduct, or an enzymes that yields a magnetic product.

In certain embodiments of this aspect, the neoplastic cell is a breastcancer cell, an ovarian cancer cell, a myeloma cancer cell, a lymphomacancer cell, a melanoma cancer cell, a sarcoma cancer cell, a leukemiacancer cell, a retinoblastoma cancer cell, a hepatoma cancer cell, aglioma cancer cell, a mesothelioma cancer cell, or a carcinoma cancercell. In certain embodiments, the neoplastic cell is a promyleocyticleukemia cell, a T lymphoblastoid cell, a breast epithelial cell, or anovarian cell. In particular embodiments, the patient is a human, such asa human patient that is suffering from a disease or disorder caused bythe presence of the neoplastic cell(s).

In another aspect, the invention provides kits for diagnosing ordetecting a neoplasm, which include at least one probe for detectingvimentin, and at least one other probe for detecting another neoplasticmarker such as nucleophosmin or HSC70. In one embodiment, the probe fordetecting vimentin is an anti-vimentin antibody or a binding fragmentthereof. In other embodiments, the probe for detecting vimentin is avimentin ligand such as modified LDL, NLK1 protein, vimentin, desmin,glial fibrillary acidic protein, and peripherin, fimbrin, RhoA-bindingkinase alpha, or protein phosphatase 2A.

In certain embodiments, the second probe for detecting a non-vimentinneoplastic marker is a nucleophosmin antibody or an HSC70 antibody. Inother embodiments, the second probe for detecting a non-vimentinneoplastic marker is a nucleophosmin ligand, such as protein kinase R(PKR), RNA, retinoblastoma protein, IRF-1, or a nuclear localizationsignals (NLS) such as the N-terminal nuclear localization signal of Rexprotein as and an HSC70 ligand. In still other embodiments, the secondprobe for detecting a non-vimentin neoplastic marker is an HSC70 ligand,such as Alzheimer's tau protein, BAG-1, small glutamine-richtetratricopeptide repeat-containing protein (SGT), (aa 642-658) ofrotavirus VP5 protein, auxilin, or the immunosuppressant5-deoxyspergualin (DSG).

In particularly useful embodiments, the kit includes a first probe whichdetects vimentin present on the surface of the test cell if it isneoplastic, and a second probe which detects another (non-vimentin)marker present of the surface of the test cell if it is neoplastic.

In yet another aspect, the invention provides a cell surfacevimentin-targeted agent for treating a cancerous neoplastic cell growth.The cell surface vimentin-targeted agent generally includes a vimentinbinding component and a therapeutic component. The vimentin bindingcomponent targets the therapeutic component to the neoplastic cellgrowth and thereby treats the cancer. The vimentin binding component andthe therapeutic component, therefore, act together such that thevimentin binding component targets the therapeutic component to theneoplasm to treat the neoplasm.

In certain embodiments, the vimentin binding component is ananti-vimentin antibody. In other embodiments, the vimentin bindingcomponent is a vimentin ligand such as LDL, NLK1 protein, vimentin,desmin, glial fibrillary acidic protein, peripherin, fimbrin,RhoA-binding kinase alpha, or protein phosphatase 2A. In particularembodiments, the vimentin binding component is a natural ligands,synthetic small molecules, chemicals, nucleic acids, peptides orprotein.

In certain useful embodiments of this aspect of the invention, thetherapeutic component is a chemotherapeutic agent such as Actinomycin,Adriamycin, Altretamine, Asparaginase, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide, Fludarabine,Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide,Imatinib, Irinotecan, Lomustine, Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,Vinblastine, Vincristine, or Vinorelbine. In particular embodiments, thetherapeutic component is in a liposome formulation.

In other embodiments, the therapeutic component is a radioisotope suchas ⁹⁰Y, ¹²⁵I, ¹³¹I, ²¹¹At or ²¹³Bi.

In still other embodiments, the therapeutic component is a toxin capableof killing or inducing the killing of the targeted multi-drug resistantneoplastic cell. Such toxins for use in the invention includePseudomonas exotoxin, diphtheria toxin, plant ricin toxin, plant abrintoxin, plant saporin toxin, plant gelonin toxin and pokeweed antiviralprotein.

In particularly useful embodiments, the vimentin binding component ofthe cell surface vimentin-targeted therapeutic agent binds to thesurface of the target cell, and the therapeutic element is internalizedand arrests growth of the cell, compromises viability of the cell orkills the cell.

In another aspect, the invention provides a method of treating aneoplasm in a subject by administering any of the cell surfacevimentin-targeted therapeutic agents described above. In certainembodiments of this aspect, the neoplasm is a breast cancer, an ovariancancer, a myeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, aretinoblastoma, a hepatoma, a glioma, a mesothelioma, or a carcinoma. Infurther embodiments, the neoplasm is from a tissue such as blood, bonemarrow, spleen, lymph node, liver, thymus, kidney, brain, skin,gastrointestinal tract, eye, breast, prostate, or ovary. In particularembodiments, the subject is a human patient, such as a human patientsuffering from a disease or disorder caused by the presence of theneoplasm.

In yet another aspect, the invention provides vaccines for treating orpreventing a neoplasm. These vaccines of the invention include avimentin polypeptide, or vimentin polypeptide subsequence thereof, andat least one pharmaceutically acceptable vaccine component. In certainembodiments, the vimentin polypeptide or polypeptide subsequence is ahuman vimentin polypeptide sequence having an amino acid sequence of SEQID NO: 1. In particular embodiments, the vimentin polypeptidesubsequence is at least eight amino acids long, and, in certainembodiments, functions as a hapten.

In certain embodiments, the vaccine formulation includes an adjuvant orother pharmaceutically acceptable vaccine component. In particularembodiments, the adjuvant is aluminum hydroxide, aluminum phosphate,calcium phosphate, oil emulsion, a bacterial product, whole inactivatedbacteria, an endotoxins, cholesterol, a fatty acid, an aliphatic amine,a paraffinic compound, a vegetable oil, monophosphoryl lipid A, asaponin, or squalene.

In another aspect, the invention provides a method of treating orpreventing a neoplasm in a subject by administering any of the vimentinvaccines described above. In certain embodiments of this aspect, theneoplasm to be treated is a breast cancer, an ovarian cancer, a myeloma,a lymphoma, a melanoma, a sarcoma, a leukemia, a retinoblastoma, ahepatoma, a glioma, a mesothelioma, or a carcinoma. In furtherembodiments, the neoplasm is from a tissue such as blood, bone marrow,spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinaltract, eye, breast, prostate, or ovary. In particular embodiments, thesubject is a human patient, such as a human patient is suffering from adisease or disorder caused by the presence of the multi-drug resistantcell.

In still another aspect, the invention provides a method for detectingdamage (e.g., pathogen infection) in a test cell by measuring a level ofcell surface-expressed vimentin protein in the test cell of a givenorigin or cell type, and comparing it to the level of cellsurface-expressed vimentin in a nondamaged cell of the same origin orcell type. If the level of cell surface-expressed vimentin in the testcell is greater than the level of cell surface-expressed vimentin in thenondamaged cell of the same given origin or cell type, then the testcell is damaged (e.g., infected).

In certain embodiments, the damaged cell is infected with a pathogen. Inparticular embodiments, the level of cell surface-expressed vimentin inthe test cell is measured by isolating a cytoplasmic membrane fractionfrom the cell and measuring the level of vimentin in the cytoplasmicmembrane fraction. In certain embodiments, the level of cellsurface-expressed vimentin in the test cell is measured with ananti-vimentin antibody. In particular embodiments, the anti-vimentinantibody measures the level of cell surface vimentin present on theintact test cell. For example, the level of antibody bound to cellsurface vimentin may be measured by immunofluorescence emission orradiolabel.

In particular embodiments, damaged cell is infected with a pathogen thatis a virus, a bacterium or a parasite. In certain embodiments, thepathogen is a virus such as HIV, West Nile virus or Dengue virus. Inother embodiments, the pathogen is a bacterium such as a Mycobacteria,Rickettsia, or Chlamydia. In still other embodiments, the pathogen is aparasite such as a Plasmodium, Leishmania, or Taxoplasma.

In certain other embodiments, the test cell is from a tissue such asblood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain,skin, gastrointestinal tract, eye, breast, prostate, or ovary. Inparticular embodiments, the test cell is from a human. In particularembodiments, the human patient is suffering from a disease or disordercaused by the presence of the pathogen infected cell.

In another aspect, the invention provides a method for detecting adamaged (e.g., pathogen-infected) cell or cells in a patient byadministering to the patient, a vimentin binding agent operably linkedto a detectable label. The label is operably linked to the vimentinbinding agent, which specifically binds to cell surface-expressedvimentin present on the damaged (e.g., pathogen-infected) cell(s) in thepatient, and is then detected, thereby locating the presence of thedamaged (e.g., pathogen-infected) (if any) in the patient. In certainembodiments, the vimentin binding agent used is an antibody or fragmentthereof. In other embodiments, the vimentin binding agent is a vimentinligand such as modified LDL, NLK1 protein, vimentin, desmin, glialfibrillary acidic protein, or peripherin, fimbrin, RhoA-binding kinasealpha, or protein phosphatase 2A. In particular embodiments, thevimentin binding agent is a natural ligand, a synthetic small molecule,a chemical, a nucleic acid, a peptide, a protein or an antibody. Inother embodiments, the detectable label is a fluorophore, a chemicaldye, a radioactive compound, a chemoluminescent compound, a magneticcompound, a paramagnetic compound, a promagnetic compound, an enzymethat yields a colored product, an enzyme that yields a chemoluminescentproduct, or an enzyme that yields a magnetic product.

In another aspect, the invention provides kits for diagnosing ordetecting pathogen infection in a test cell. The kits include one probefor the detection of vimentin and a second probe for the detection ofanother marker of damage (e.g., pathogen infection) such asnucleophosmin or HSC70. In particular embodiments, the kits includeanti-vimentin antibody as the probe for detecting vimentin. In otherembodiments, the kits include a vimentin ligand such as LDL, NLK1protein, vimentin, desmin, glial fibrillary acidic protein, andperipherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase2A. In further embodiments, the kits include a nucleophosmin antibody oran HSC70 antibody as probes for detecting the second, non-vimentindamage (e.g., pathogen infection) marker. In other embodiments, thesecond probe may be a nucleophosmin ligand (such as protein kinase R(PKR), RNA, retinoblastoma protein, IRF-1, or a nuclear localizationsignals (NLS) such as the N-terminal nuclear localization signal of Rexprotein), or an HSC70 ligand (such as Alzheimer's tau protein, BAG-1,small glutamine-rich tetratricopeptide repeat-containing protein (SGT),(aa 642-658) of rotavirus VP5 protein, auxilin, or the immunosuppressant5-deoxyspergualin (DSG)). In certain embodiments, the vimentin bindingcomponent is a natural ligand, a synthetic small molecule, a chemical, anucleic acid, a peptide, a protein, or an antibody or fragments thereof.

In yet another aspect, the invention provides cell surfacevimentin-targeted agents for treating infection by a pathogen. Thevimentin-targeted agent includes a vimentin binding component and atherapeutic component. The vimentin binding component targets thetherapeutic component to the pathogen infected cell and thereby treatsthe infection. In certain embodiments, the vimentin binding agent is ananti-vimentin antibody. In other embodiments the vimentin bindingcomponent is a vimentin ligand such as modified LDL, NLK1 protein,vimentin, desmin, glial fibrillary acidic protein, and peripherin,fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. Incertain embodiments, the vimentin binding component is a natural ligand,a synthetic small molecule, a chemical, a nucleic acid, a peptide, aprotein, or an antibody or fragments thereof.

In particular embodiments, the therapeutic component is anantibacterial, antiviral or antiparasitic agent. In certain embodiments,the vimentin binding component binds to the surface of the target celland the therapeutic element is internalized and arrests growth of thepathogen, compromises viability of the pathogen or kills thepathogen-infected cell.

In yet another aspect, the invention provides vaccines for treating orpreventing infection by a pathogen. These vaccines include a vimentinpolypeptide or polypeptide subsequence at least one pharmaceuticallyacceptable vaccine component. In certain embodiments, the vimentinpolypeptide is a human vimentin polypeptide sequence having an aminoacid sequence of SEQ ID NO: 1. In particular embodiments, the vimentinpolypeptide subsequence is at least eight amino acids long, and, incertain embodiments, functions as a hapten.

In certain embodiments, the vaccine formulation includes an adjuvant orother pharmaceutically acceptable vaccine component. In particularembodiments, the adjuvant is aluminum hydroxide, aluminum phosphate,calcium phosphate, oil emulsion, a bacterial product, whole inactivatedbacteria, an endotoxins, cholesterol, a fatty acid, an aliphatic amine,a paraffinic compound, a vegetable oil, monophosphoryl lipid A, asaponin, or squalene.

In another aspect, the invention provides a method of treating orpreventing an infection in a subject by administering any of thevimentin vaccines described above. In certain embodiments of thisaspect, the subject is a human patient. In particular embodiments thehuman patient is suffering from a disease or disorder caused by thepresence of infection. In certain embodiments, the infection is in atissue such as blood, bone marrow, spleen, lymph node, liver, thymus,kidney, brain, skin, gastrointestinal tract, eye, breast, prostate orovary.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photographic representation of a silver stained 2-D gelresolving total cell extracts from MCF-7 cells showing the presence ofvimentin as a 53 kDa spot.

FIG. 1B is a photographic representation of a silver stained 2-D gelresolving total cell extracts from multidrug resistant MCF-7/AR cellsshowing the presence of vimentin as a 53 kDa spot.

FIG. 2A is a representation of an anti-vimentin immunoblot of total cellextracts from MCF-7 cells resolved by 2-D gels and transferred ontonitrocellulose.

FIG. 2B is a representation of an anti-vimentin immunoblot of total cellextracts from multidrug resistant MCF-7AR cells resolved by 2-D gels andtransferred onto nitrocellulose.

FIG. 3A is a representation of a streptavidin-HRP blot of surfacebiotinylated total cell extracts from CEM cells resolved by 2-D gels andtransferred onto nitrocellulose.

FIG. 3B is a representation of a streptavidin-HRP blot of surfacebiotinylated total cell extracts from multidrug resistant CEM/VLB cellsresolved by 2-D gels and transferred onto nitrocellulose.

FIG. 4A is a photographic representation of a Western blot analysis of10% SDS-polyacrylamide gel-resolved biotinylated total cell extractsprepared from HL60 and HL60/AR cells using an anti-vimentin antibody.

FIG. 4B is a photographic representation of a Western blot analysis of10% SDS-polyacrylamide gel resolved-biotinylated total cell extractsprepared from streptavidin purified extracts prepared from surfacebiotinylated total cell extracts and using an anti-vimentin antibody.

FIG. 4C is a photographic representation of a Western blot analyses of10% SDS-polyacrylamide gel resolved-biotinylated total cell extractsprepared from anti-vimentin immunoprecipitates of surface biotinylatedtotal cell extracts and using an anti-vimentin antibody.

FIG. 4D is a photographic representation of a Western blot analysis of10% SDS-polyacrylamide gel resolved-biotinylated total cell extractsprepared from anti-vimentin immunoprecipitates of surface biotinylatedtotal cell extracts using a streptavidin-HRP probe.

FIG. 5A is a photographic representation of a Western blot analysis of10% SDS-polyacrylamide gel resolved biotinylated total cell extractsprepared from MCF-7 and MCF-7/AR cells using an anti-vimentin antibody.

FIG. 5B is a photographic representation of a Western blot analysis of10% SDS-polyacrylamide gel resolved biotinylated total cell extractsprepared from streptavidin purified extracts prepared from surfacebiotinylated total cell extracts and using an anti-vimentin antibody.

FIG. 5C is a photographic representation of Western blot analyses of 10%SDS-polyacrylamide gel resolved biotinylated total cell extractsprepared from anti-vimentin immunoprecipitates of surface biotinylatedtotal cell extracts and using an anti-vimentin antibody.

FIG. 5D is a photographic representation of Western blot analyses of 10%SDS-polyacrylamide gel resolved biotinylated total cell extractsprepared from anti-vimentin immunoprecipitates of surface biotinylatedtotal cell extracts and using an HRP-streptavidin probe.

FIG. 6 is a graphic representation of the results of FACS analysis forthe surface expression of vimentin using polyclonal anti-vimentinantibody on HL60 (white bar) and HL60/AR (gray bar) cell lines.

FIG. 7 is a graphic representation of the result of FACS analysis forthe surface expression of vimentin at saturating amounts of monoclonalanti-vimentin antibody on HL60 (white bar) and HL60/AR (gray bar) celllines.

FIG. 8 is a graphic representation of the results of FACS analysis forthe surface expression of vimentin using monoclonal anti-vimentinantibody on Molt4 (white bars) and Molt4/DOX (gray bars) cell lines.

FIG. 9 is a graphic representation of the results of FACS analysis forthe surface expression of vimentin using monoclonal anti-vimentinantibody on breast cancer sensitive and drug-resistant model cell lines.

FIG. 10 is a graphic representation of the results of FACS analysis forthe surface expression of vimentin using monoclonal anti-vimentinantibody on MDA drug-sensitive and MDA/MITO drug-resistant cell lines.

FIG. 11 is a graphic representation of the results of quantitative FACSanalysis of the surface expression of vimentin on MCF-7 and MCF-7/ARcell lines.

FIG. 12A is a schematic representation of the polypeptide sequence of ahuman vimentin corresponding to GenBank Accession No. P08670 (SEQ ID NO.1).

FIG. 12B is a schematic representation of the nucleotide sequence of ahuman vimentin-encoding nucleic acid sequence corresponding to GenBankAccession No. X56134 (SEQ ID NO. 2). The initiation and terminationcodons of the vimentin protein open reading frame are underlined.

FIG. 13A is a flow chart showing a procedure for immunohistochemicalstaining of non-permeabilized adherent tumor cells.

FIG. 13B is a flow chart showing a procedure for immunohistochemicalstaining of non-permeabilized adherent tumor cells.

FIG. 14 is a photographic representation of permeabilized andnon-permeabilized MCF-7 and MCF-7/AR cells immunostained withanti-vimentin antibody (clone V9, NeoMarker MS-129-P) using theprocedure described in FIGS. 13A&B. Mouse IgG1 was used as negativeisotype matching control and didn't show any staining (not shown). OnlyMCF-7/AR shows surface exposed Vimentin.

FIG. 15 is a graphic representation of the results of the intact cellradioimmunoassay for the surface exposure of vimentin in HL60 drugsensitive and HL60/AR drug-resistant cell lines. CD33 was used aspositive control for surface exposure

FIG. 16 is a graphic representation of the results of the cytotoxicityof radiolabeled anti-vimentin on HL60 and HL60/AR cells. CD33 was usedas positive control for surface exposure

FIG. 17A is a table showing the results of 5 independent Scatchardanalyses of the surface exposure of vimentin in MDA/mito cells. R²values represent the quality of the non-linear curve fit used todetermine K_(d) and Bmax. The analysis was performed with PrizmGraphPad.

FIG. 17B is a representation of the Scatchard analysis performed in thefirst experiment reported in table 17A.

FIG. 17C is a table reporting the number of molecules of vimentinpresent on the surface of various breast and ovarian cell lines. R/Srepresents the folds overexpression of surface vimentin when comparingsensitive and resistant counterpart cells.

FIG. 18 is a graphic representation of the results of the intact cellradioimmunoassay for the induction of surface expression of vimentinwith doxorubicin, taxol, and mitoxanthrone.

FIG. 19A is a graphic representation of the results of the assessment ofthe internalization of vimentin for MCF-7/AR cells kept in suspensionfor the duration of the experiment.

FIG. 19B is a graphic representation of the results of the assessment ofthe internalization of vimentin for adherent MCF-7/AR, MDA, MDA/AR andMDA/mito cells.

4. DETAILED DESCRIPTION

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issuedU.S. patents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, that are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

In particular, this application incorporates the following patentapplications by reference in their entirety: U.S. Ser. No. 60/433,480,filed Dec. 13, 2002 and entitled “Vimentin Detection-Based Methods forDiagnosing and Treating Damaged Cells, Neoplastic Cells and MultidrugResistance;” U.S. Ser. No. 60/433,351, filed Dec. 13, 2002 and entitled“Nucleophosmin Detection-Based Methods for Diagnosing and TreatingDamaged Cells, Neoplastic Cells and Multidrug Resistance”, as well asU.S. Ser. No. ______, filed Dec. 15, 2003 and entitled “NucleophosminDirected Diagnostics and Therapeutics for Multidrug Resistant NeoplasticDisease;” and U.S. Ser. No. 60/438,012, filed Jan. 1, 2003 and entitled“HSC70 Detection-Based Methods for Diagnosing and Treating DamagedCells, Neoplastic Cells and Multidrug Resistance,” as well as U.S. Ser.No. ______, filed Dec. 15, 2003 and entitled “HSC70 DirectedTherapeutics and Diagnostics for Multidrug Resistant NeoplasticDisease.”

4.1 General

The invention provides methods and reagents for diagnosing, preventingand/or treating cancer, and for diagnosing, preventing and/or treatingthe development of both naturally occurring and drug-induced MDRphenotypes of both non-cancerous and cancerous cells. The inventionallows for improvement of the clinical management of multidrug resistanttumors and pathogen infections. Moreover, the invention provides areagent that allows the identification of patients having neoplastic ordamaged cells, including MDR cells, thus allowing improvements in thetreatment, monitoring, diagnosis, and medical imaging of multidrugresistant cancer and pathogen infections.

Accordingly, an embodiment of the invention provides a method fordetecting multidrug resistance in a test damaged cell. The methodincludes measuring the level of cell surface expression of vimentinprotein damaged cell of a specific cell type; measuring the level ofcell surface-expressed vimentin protein on a drug susceptible damagedcell of the same cell type; and determining that the test damaged cellis multidrug resistant if an increased level of cell surface-expressedvimentin is present compared to the level of cell surface-expressedvimentin present on the drug-susceptible damaged cell. In particularembodiments, the level of cell surface-expressed vimentin is measured byseparating the cellular components of the test damaged cell and the drugsusceptible damaged cell into fractions, and measuring the level ofvimentin in the cellular component fraction containing the cytoplasmicor plasma membrane of the cells.

In some embodiments, the test damaged cell is infected with a pathogen.In particular embodiments, the pathogen is a virus, a bacterium, or aparasite. Exemplary viruses include, but are not limited to, HIV, WestNile virus and Dengue virus. Exemplary bacteria include, but are notlimited to, Mycobacteria, Rickettsia, and Chlamydia. Exemplary parasitesinclude, but are not limited to Plasmodium, Leishmania, and Taxoplasma.In some embodiments, the test damaged cell is from a tissue selectedfrom the group consisting of blood, bone marrow, spleen, lymph node,liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast,prostate and ovary. In certain embodiments, the test damaged cell isfrom a human.

The invention also provides a method for detecting multidrug resistancein a test neoplastic cell. The method includes measuring the level ofcell surface-expressed vimentin protein on the test neoplastic cell of aspecific cell type; measuring the level of cell surface-expressedvimentin protein on a drug susceptible neoplastic cell of the same type;and determining that the test neoplastic cell is multidrug resistant ifan increased level of cell surface expression of vimentin is presentcompared to the level of cell surface expression present on the drugsusceptible neoplastic cell. In particular embodiments, the level ofcell surface expression of vimentin is measured by separating thecellular components of the test neoplastic cell and the drug susceptibleneoplastic cell into fractions, and measuring the level of vimentin inthe cellular component fraction containing the cytoplasmic or plasmamembrane of the cells.

Exemplary neoplastic cells include, but are not limited to, lymphomacells, melanoma cells, sarcoma cells, leukemia cells, retinoblastomacells, hepatoma cells, myeloma cells, glioma cells, mesothelioma cellsand carcinoma cells. In certain embodiments, the test neoplastic cell isfrom a tissue selected from the group consisting of blood, bone marrow,spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinaltract, eye, breast, prostate and ovary. In certain embodiments, the testneoplastic cell is from a human.

The invention further provides a method for detecting a multidrugresistant cell in a patient. The method includes administering a bindingagent that specifically binds to vimentin protein operably linked to adetectable label, and detecting increased binding of the binding agentspecifically bound to vimentin protein on the surface of a multidrugresistant cell in the patient compared to the amount of binding agentbound to vimentin protein on the surface of a drug susceptible cell. Insome embodiments of the invention, a medical imaging device or systemdetects the binding agent specifically bound to the cell surface of amultidrug resistant cell in the patient. Exemplary binding agentsinclude, but are not limited to, natural ligands, synthetic smallmolecules, chemicals, nucleic acids, peptides, proteins, antibodies andfragments thereof. In certain embodiments, the binding agent is anantibody.

Exemplary detectable labels include, but are not limited to,fluorophores, chemical dyes, radioactive compounds, chemoluminescentcompounds, magnetic compounds, paramagnetic compounds, promagneticcompounds, enzymes that yield a colored product, enzymes that yield achemoluminescent product and enzymes that yield a magnetic product. Incertain embodiments, the patient is a human. In some embodiments, themultidrug resistant cell is a damaged cell or a neoplastic cell. Incertain embodiments, the damaged cell is infected with a pathogen.Exemplary pathogens include, but are not limited to, viruses, bacteriaand parasites. Exemplary viruses include, but are not limited to, HIV,West Nile virus and Dengue virus. Exemplary bacteria include, but arenot limited to, Mycobacteria, Rickettsia, and Chlamydia. Exemplaryparasites include, but are not limited to, Plasmodium, Leishmania, andTaxoplasma. In particular embodiments, the neoplastic cell is selectedfrom the group consisting of a breast cancer cell, an ovarian cancercell, a myeloma cancer cell, a lymphoma cancer cell, a melanoma cancercell, a sarcoma cancer cell, a leukemia cancer cell, a retinoblastomacancer cell, a hepatoma cancer cell, a glioma cancer cell, amesothelioma cancer cell, or a carcinoma cancer cell. In particularembodiments, the patient is a human. In some embodiments, the patient issuffering from a disease or disorder caused by the presence of themultidrug resistant cell.

The invention also provides a method for detecting a neoplastic cell.The method includes measuring the cell surface-expressed vimentinprotein on the suspected neoplastic cell and determining that the cellis neoplastic if an increased level of cell surface-expressed vimentinprotein is present compared to the level of cell surface-expressedvimentin present on a normal cell of the same type. In certainembodiments, cell surface-expressed vimentin is measured by separatingthe cellular components of the suspected neoplastic cell and the normalcell into fractions, and measuring the level of vimentin in the cellularcomponent fraction containing the cytoplasmic or plasma membrane of saidcells. In some embodiments, the cellular components of the test andnormal cells are contacted with a detectable binding agent, followed bydetection of the binding agent to determine the level of cellsurface-expressed vimentin protein on each cell. In other embodiments,cell surface-expressed vimentin is measured by contacting an intact,suspected neoplastic cell of a specific cell type and an intact normalcell of the same type cell with a detectable binding agent thatspecifically binds to vimentin protein, and detecting the binding of theagent to determine the level of cell surface-expressed vimentin proteinon the intact cells.

In certain embodiments, the test cell is from a tissue selected from thegroup consisting of blood, bone marrow, spleen, lymph node, liver,thymus, kidney, brain, skin, gastrointestinal tract, eye, breast,prostate and ovary. In a particular embodiment, the suspected neoplasticcell is from a human. In one embodiment, the human is suffering from acancer caused by the presence of the neoplastic cell.

As used herein, a “neoplastic cell” is a cell that shows aberrant cellgrowth, such as increased cell growth. A neoplastic cell may be ahyperplastic cell, a cell that shows a lack of contact inhibition ofgrowth in vitro, a tumor cell that is incapable of metastasis in vivo,or a cancer cell that is capable of metastasis in vivo. Non-limitingexamples of neoplastic cells include lymphoma cells, melanoma cells,breast cancer cells, ovarian cancer cells, prostate cancer cells,sarcoma cells, leukemic cells, retinoblastoma cells, hepatoma cells,myeloma cells, glioma cells, mesothelioma cells, and carcinoma cells.

As used in accordance with the invention, a “damaged cell” means a cellthat is non-neoplastic, but that has been otherwise injured. Forexample, the non-neoplastic damaged cell may be a cell infected with apathogen, such as a virus, a bacterium, or a parasite. In onenon-limiting example, the cells may be damaged by infection with amulti-cellular parasite, or damaged by the effects of infection by aparasite. Such non-limiting parasites include schistosomes, plasmodiums,trypanosomes, Leishmania, and Taxoplasma.

As used herein, the terms, “multidrug resistant” and “multidrugresistance,” are used to refer to the development, in a neoplastic cellor damaged cell, of resistance to a number of different drugs, includingdrugs to which the neoplastic cell or damaged cell was never exposed.For example, if a patient suffering from leukemia being treated withvincristine develops leukemia cells resistant to vincristine as well asother chemotherapeutics that the patient had never received (e.g.,methotrexate or mercaptopurine), that patient's leukemic cells aremultidrug resistant. Similarly, if a patient suffering from tuberculosisbeing treated with penicillin develops tuberculosis-infected cellsresistant to penicillin as well as other drugs that the patient hadnever received (e.g., erythromycin), that patient'stuberculosis-infected cells are multidrug resistant. Notably, multidrugresistance (MDR) may include acquired simultaneous resistance to a widespectrum of drugs, including drugs with little structural or evenfunctional similarity to the original drug(s), and results in reducedefficacy of all the drugs concerned.

Note that the terms, “multidrug resistant” and “multidrug resistance,”are used to describe a neoplastic cell or a damaged cell that ismultidrug resistant due to either the classical mechanism (i.e.,involving P-glycoprotein or another MDR protein) or an atypicalmechanism (non-classical mechanism) that does not involve P-glycoprotein(e.g., an atypical mechanism that involves the MRP1 multidrug resistancemarker).

As used herein, the term “MDR protein” includes any of several integraltransmembrane glycoproteins of the ABC type that are involved in(multiple) drug resistance. These include MDR 1 (P-glycoprotein orP-glycoprotein 1), an energy-dependent efflux pump responsible fordecreased drug accumulation in multidrug resistant cells. Examples ofMDR 1 include human MDR 1 (see, e.g., database code MDR1_HUMAN, GenBankAccession No. P08183, 1280 amino acids (141.34 kDa)). Other MDR proteinsinclude MDR 3 (or P-glycoprotein 3), which is an energy-dependent effluxpump that causes decreased drug accumulation but is not capable ofconferring drug resistance by itself. Examples of MDR 3 include humanMDR 3 (see, e.g., database code MDR3_HUMAN, GenBank Accession No.P21439, 1279 amino acids (140.52 kDa). Other MDR-associated proteinsparticipate in the active transport of drugs into subcellularorganelles. Examples from human include MRP 1, MultidrugResistance-associated Protein 1, database code MRP HUMAN, GenBankAccession No. P33527, 1531 amino acids (171.47 kDa).

In accordance with the invention, a cell (e.g., a neoplastic or damagedcell) that develops multidrug resistance can develop such MDR statuseither by being exposed to a drug (e.g., a chemotherapeutic drug or anantibiotic drug), or by naturally developing such MDR (i.e., withouthaving been exposed to the drug to which the cell has developedresistance). In this respect, the invention allows the detection of thepotentially multidrug resistant character of a neoplasm even before theneoplasm has been treated. Similarly, the invention allows for theeffective treatment of, for example, potentially multidrug resistantneoplasms even before the neoplasm has been treated and shown to be drugresistant.

The invention also allows the early identification of patients havingsuch MDR neoplastic or damaged cells. For example, where the patientidentified as having such cells is an asymptomatic patient who is beingtreated for an infectious disease, or had received treatment for aninfectious disease (e.g., hepatitis B), the invention allowsidentification of these patients prior to resurgence of symptoms, aswell as allows the monitoring of these patients during treatment with adrug, such that the treatment regimen can be altered if such MDR cellsare detected. Similarly, where the patient identified as having suchcells is a patient in remission of cancer or is being treated for cancer(e.g., a patient suffering from breast cancer, ovarian cancer, prostatecancer, leukemia, etc.), the invention allows identification of thesepatients prior to resurgence and/or progression of their cancer, as wellas allows the monitoring of these patients during treatment with a drug,such that the treatment regimen can be altered.

The invention stems from the discovery that cell surface expressedvimentin is expressed on the surface of multidrug resistant cells atmeasurably higher levels than that found on other cells and, thus, isuseful in part as a marker for multidrug resistance of a cell. Animportant advantage of the vimentin protein cell surface marker is thatvimentin is present only in negligible levels on the surface of normalcells of the body. This expression is in contrast to the situation withother known MDR markers such as P-glycoprotein and the multidrugresistance protein (MRP), which are present at variable levels on thesurface of cells of different normal tissues, including high levels onthe surface of liver, kidney, stem cells, and blood-brain barrierepithelial cells (Cordon-Cardo C. et al., J. Histochem. Cytochem. 38:1277-1287, 1990; Nakamara T. et al., Drug Metabatabolism & Disposition,30: 4-6, 2002). As a consequence, use of agents directed against MDRcancer cell markers such as P-glycoprotein and MRP, have been limited byside effects caused by the killing of normal cells that also expresshigh levels of cell surface P-glycoprotein and MRP (see, e.g.,FitzGerald, D. J. et al., Proc. Natl. Acad. Sci. 84: 4288-4292, 1987).The present invention overcomes this detrimental side effect because thevimentin MDR marker is expressed on the cell surface of drug-sensitivecancer cells at moderate levels (as compared to the very low tonegligible levels on drug-sensitive non-cancerous normal cells), and athigher levels on MDR cancer cells and MDR non-cancerous damaged cells(e.g., cells infected with a virus). Thus, the invention provides agentsdirected toward cell surface-expressed vimentin which kills MDR-damagedor MDR-neoplastic cells as well as drug-sensitive neoplastic cells,while leaving normal cells unscathed.

It should be noted that the same MDR neoplastic or damaged cell mayexpress more than one MDR marker (e.g., may express both P-glycoproteinand vimentin) simultaneously, or may express these MDR markersindependently. This joint expression on the same MDR cell offers thepossibility of combining binding agents directed against more than onecell surface MDR marker. For example, a sub-lethal dosage of a bindingagent that specifically binds to vimentin can be combined with asub-lethal dosage of a binding agent that specifically binds toP-glycoprotein. Since normal cells do not express vimentin on their cellsurface, they will not be harmed by the binding agent that specificallybinds to vimentin. Rather, only MDR cells that express bothP-glycoprotein and vimentin on their cell surface will be killed by thiscombination therapy.

Thus, cell surface-expressed vimentin is a superior marker for use intherapies (such as immunotoxin therapy) that kill MDR neoplastic cellsand MDR damaged cells bearing the vimentin marker on their cell surface,since normal cells would be spared from cell killing, thus reducing oreliminating harmful side effects of treatment. Similarly, diagnosis andimaging of MDR neoplastic or damaged cells using the cell surfacevimentin marker are more sensitive and accurate, and provide fewer falsepositives as compared to diagnosis and imaging of MDR neoplastic anddamaged cells using the other MDR markers such as P-glycoprotein or MRPthat are also expressed on normal tissues. Moreover, cell surfacevimentin is useful as an anti-MDR cancer vaccine antigen or an anti-MDRdamaged cell vaccine antigen for vaccination of patients against theircancers or damaged cell tissue expressing vimentin on their cellsurface.

The invention also allows the identification of those patients whosedamaged or neoplastic cells have acquired multidrug resistance. In somesituations, the patient is identified when he/she no longer responds tothe drug being used in his/her treatment. For example, a breast cancerpatient in remission being treated with a chemotherapeutic agent (e.g.,vincristine) may suddenly come out of remission, despite beingconstantly treated with the chemotherapeutic agent. Unfortunately, sucha patient is often found also to be unresponsive to otherchemotherapeutic agents, including some to which the patient has neverbeen exposed. Of course, after these patients become multidrugresistant, treating these patients to control their now-resurgent canceror disease caused by a damaged cell is difficult and may require moredrastic therapies, such as radiotherapy or surgery (e.g., bone marrowtransplantation or amputation of necrotic tissue).

The invention allows an early diagnosis of multidrug resistance bydetecting increased amounts of cell surface expression of vimentin onthe neoplastic or damaged cells of the patient. Such an early diagnosisallows patients who are initially drug responders and sensitive to drugtreatment to be distinguished from those who are initially drugnon-responders. Further, diagnostic procedures using vimentin cellsurface expression may also be used to follow the development andemergence of MDR damaged or neoplastic cells that are resistant to thetreatment drug and that arise during the course of drug treatment. Forexample, such procedures are useful for treating AIDS patients that havebeen treated with AZT and that have been reported to subsequentlydevelop multidrug resistance to a wide spectrum of anti-viral,antibacterial, and anticancer drugs (see Gollapudi et al., Biochem.Biophys. Res. Commun. 171: 1002-1007, 1990; Antonelli et al., AIDSResearch and Human Retroviruses 8: 1839-1844, 1992).

In addition, diagnostic assays for vimentin cell surface expression areuseful for selecting patients in clinical studies involving therapy fortreatment of damaged or neoplastic cells. Hence, the presence ofvimentin on the cell surface of a patient's cells either qualifies ordisqualifies that patient from being included in a given clinical study.

Accordingly, in one aspect, the invention provides a method fordetecting multidrug resistance in a test damaged cell suspected of beingmultidrug resistant. The method includes measuring the level of cellsurface-expressed vimentin protein on a test damaged cell of a specificcell type; measuring the level of cell surface-expressed vimentinprotein on a drug susceptible damaged cell of the same type; anddetermining that the test damaged cell is multidrug resistant if anincreased level of cell surface-expressed vimentin protein is presentcompared to the level of cell surface-expressed the vimentin proteinpresent on the non-MDR damaged cell (e.g., a damaged cell that issusceptible to a drug).

In another aspect, the invention provides a method for detectingmultidrug resistance in a test neoplastic cell suspected of beingmultidrug resistant. The method includes measuring the level of cellsurface-expressed vimentin protein on a test neoplastic cell of aspecific cell type; measuring the level of cell surface-expressedvimentin on a drug susceptible neoplastic cell of the same type; anddetermining that the test cell is multidrug resistant if an increasedlevel of cell surface-expressed vimentin protein is present compared tothe level of cell surface-expressed vimentin present on the drugsusceptible neoplastic cell. In some embodiments, the test neoplasticcell or test damaged cell expresses an amount of vimentin on its cellsurface that is at least two-fold higher than the level of cell surfaceexpression of the vimentin protein on a normal cell or on a non-MDRneoplastic cell or non-MDR damaged cell, respectively. Such adetermination of level of cell surface-expressed vimentin can be made byany number of known methods including, without limitation, those methodsdescribed below.

As described below, although all normal cells express the vimentinprotein intracellularly, it was unexpectedly discovered that neoplasticcells and damaged cells express the full length vimentin protein ontheir cell surface. Moreover, as these neoplastic cells or damaged cellsbecome multidrug resistant, they express increased amounts of the fulllength vimentin protein on their cell surface. Drug-sensitive normalcells contain vimentin protein mainly in their nucleus, nucleolus, andcytoplasm. A moderate amount of vimentin protein is also expressed onthe surface of drug-sensitive neoplastic cells. Drug-sensitive cellsthat do not express vimentin on their cell surface (or that express asmall amount of vimentin at their cell surface) are killed by treatmentwith a drug. In contrast, neoplastic or damaged cells that acquiremultidrug resistance, whether naturally or after treatment with a drug,are recognizable because they express elevated levels of vimentinprotein on their cell surface. Thus, antibodies or other binding agentsthat specifically bind to cell-surface vimentin protein are useful fordeveloping methods for diagnosis, treatment, screening, and imaging ofMDR damaged cells and MDR neoplastic cells that express vimentin ontheir cell surface.

In certain embodiments, the test damaged cell is from a tissue, forexample, from a biopsy of damaged tissue (e.g., necrotic tissue), orfrom a type of cell that is infected by the pathogen. For example, thehepatitis B virus typically infects only liver cells; thus, a damagedcell (i.e., a liver cell infected by hepatitis B virus) is from a tissue(i.e., liver). Similarly, the Human Immunodeficiency Virus (HIV)typically infects only CD4+ T cells and macrophages; thus a damaged cell(e.g., a CD4+ T cell infected with HIV) is from a tissue (i.e., blood orbone marrow).

Note that in some limited situations, infection by a virus may cause acell to become neoplastic. For example, some B cells, when infected withthe Epstein Barr Virus (EBV), become neoplastic. Such a neoplastic Bcell, although damaged by virtue of its infection with a virus, isincluded herein as a “neoplastic cell”, not a damaged cell.

In certain embodiments, the test neoplastic cell is from a tissue, forexample, from a biopsy of a hyperplastic tissue (e.g., a lump in thebreast). Non-limiting examples of tissues from which a test neoplasticcell can be from include blood, bone marrow, spleen, lymph node, liver,thymus, kidney, brain, skin, gastro-intestinal tract, eye, breast,prostate, and ovary.

In accordance with the invention, the neoplastic cell may be from apatient, such as a human, who is suffering from a disease or disorderwhere the disease or disorder is caused by the presence of theneoplastic cell. For example, where the neoplastic cell is a neoplasticmelanoma cell, the disease is a cancer of the melanoma cell (i.e., thecancer is melanoma which is caused by aberrant cell growth andmetastasis of the neoplastic cell).

In accordance with the invention, the damaged cell may be from apatient, such as a human, who is suffering from a disease or disorderwhere the disease or disorder is caused by the presence of the damagedcell. For example, where the damaged cell is infected with a pathogen,the disease is an infection caused by the presence of those damagedcells infected by the pathogen or lack thereof (e.g., AIDS caused by thelack of CD4⁺ T cells which were infected by the HIV virus).

As used herein, a “patient suffering from a disease or disorder” means apatient who has the clinical manifestations and/or symptoms of a diseaseor disorder. In certain situations, a patient with a disease or disordermay be asymptomatic, and yet still have clinical manifestations of thedisease or disorder. For example, a patient suffering from leukemia, maynot be symptomatic (e.g., may not be sick or weak), but shows theclinical manifestation in that the patient has a larger number of whiteblood cells as compared to a healthy individual of the same age andweight. In another non-limiting example, a patient suffering frominfection with a virus (e.g., HIV), may not be symptomatic (e.g., maynot show a diminished CD4+ T cell count), but shows the clinicalmanifestation in that the patient has anti-HIV antibodies.

According to the invention, those damaged cells or neoplastic cells thathave become multidrug resistant are distinguishable from those cellsthat are not multidrug resistant by the increased expression of the fulllength vimentin protein on the cell surface of multidrug resistantcells. Representative nucleotide and amino acid sequences of vimentinare set forth in FIG. 12. Thus, when the cellular components areseparated into fractions, those cells that are multidrug resistantcontain vimentin in their cytoplasmic membrane fraction. Cell surfaceexpression of vimentin protein may also be routinely detected bynon-limiting methods such as FACS analysis, cell surface biotinylationfollowed by 2-D gels, immunoprecipitation (see Examples), orimmunofluorescent analysis of fixed clinical specimens, and other typesof routine methods performed by those skilled in the art.

In some embodiments, measuring the level of expression of a vimentinprotein on the surface of the test damaged cell or test neoplastic cellcomprises separating the cellular components of the test cell intofractions and then examining the fraction containing the cytoplasmic orplasma membrane for vimentin.

Alternatively, the measuring step comprises separating the products ofenzymatic digestion of cell surface expressed vimentin protein from thetest cell. The resulting peptides from the digested surface exposedproteins are isolated by quickly spinning the cells and leaving thedigested peptides in the supernatant. These digested peptides are thenanalyzed by various methods (e.g., immunological methods or massspectroscopy) to determine if they are vimentin peptides.

Separation of cellular components may be performed by any standardseparation procedure including, without limitation, thin layerchromatography, gas chromatography, high performance liquidchromatography, paper chromatography, affinity chromatography,supercritical flow chromatography, gel electrophoresis, and theprocedures described below in the Examples section. Separationprocedures are generally known (see, e.g., Scopes and Scopes, ProteinPurification: Principles and Practice, Springer Verlag 1994).

In some embodiments, measuring the level of expression of a vimentinprotein on the surface of the test damaged cell includes contacting theintact test damaged cell or test neoplastic cell with a detectablebinding agent that specifically binds to a vimentin protein. In someembodiments, the cell is then fractioned and then amount of labeledvimentin in the fraction containing the cytoplasmic or plasma membraneis then measured.

The binding agents useful in the present invention are any whichspecifically bind to a vimentin protein or fragment thereof. The bindingagent can specifically bind to any portion of the vimentin protein sincethe entire protein is expressed on the cell surface of multidrugresistant damaged or neoplastic cells. The binding agent of theinvention specifically binds to the surface of a neoplastic or damagedcell expressing vimentin on its cell surface at high levels compared tothat amount expressed in the surface of a drug-susceptible neoplastic ordrug-susceptible damaged cell, thereby identifying that cell as beingmultidrug resistant.

Of course, since vimentin is also expressed inside of normal cells,drug-sensitive neoplastic cells, and drug-sensitive damaged cells, ifsuch normal cells and drug-sensitive neoplastic or damaged cells arefirst lysed or if their membranes are permeabilized prior to addition ofthe binding agent, the binding agent will also bind to intracellularvimentin in normal and drug-sensitive neoplastic or damaged cells.

In some embodiments, MDR neoplastic cells or MDR damaged cells thatexpress vimentin on their cell surface are distinguishable from othertypes of cells, in that the MDR neoplastic or damaged cells express thevimentin protein on their cell surface at levels that are at leasttwo-fold higher than the normal cells from the tissue of origin of theneoplastic or damaged cells, or at levels that are at least two-foldhigher than drug-sensitive neoplastic or damaged cells from the tissueof origin. For example, a leukemic T cell expresses more vimentin on itscell surface than a normal T cell. Moreover, as described below, a MDRleukemic T cell expresses at least twice as much vimentin on its cellsurface as a leukemic T cell that is not multidrug resistant. Similarly,as described in the examples below, an MDR breast cancer cell expressesat least twice as much vimentin on its cell surface as itsdrug-sensitive counterpart (i.e., the drug-sensitive counterpart is notmultidrug resistant). Thus, when the cellular components are separated(e.g., into cytoplasmic membrane fraction and cytosolic fraction), thoseMDR neoplastic or damaged cells that express vimentin on their cellsurface contain vimentin in their cytoplasmic membrane fractions at twofold or higher levels than do other cells from the same tissue that arenot multidrug resistant, regardless whether or not the non-multidrugresistant cell is normal, neoplastic, or damaged.

In another aspect, the invention provides a binding agent thatspecifically binds to a vimentin protein or fragment thereof. As usedherein, “specifically binds” means that a binding agent recognizes andbinds to a vimentin protein or fragment thereof, but does notsubstantially recognize and bind to other molecules in a sample. Thus, abinding agent of the invention specifically binds to the surface of aMDR cell that expresses vimentin on its cell surface. A useful bindingagent forms an association with the vimentin protein with an affinity ofat least 10⁶M⁻¹, or at least 10⁷M⁻¹, or at least 10⁸M⁻¹, or at least10⁹M⁻¹ either in water, under physiological conditions, or underconditions which approximate physiological conditions with respect toionic strength, e.g., 140 mM NaCl, 5 mM MgCl₂.

As used herein, a “binding agent” is a molecule that specifically bindsor attaches to a vimentin protein or fragment thereof. A binding agentneed not be any particular size or have any particular structure so longas it specifically binds to the vimentin protein or fragment thereof.Thus, a “binding agent” is a molecule that specifically binds to orattaches to any region (e.g., three dimensional structure, amino acidsequence, or particular small chemical groups) so long as itspecifically binds to the vimentin protein or fragment thereof.Non-limiting examples of binding agents include natural ligands (such ashormones or GTP), as well as synthetic small molecules, chemicals,nucleic acids, peptides, and proteins such as hormones, antibodies, andportions thereof. Typically, the binding agent's ability to specificallybind an epitope is based on highly complementary structures. That is,the shape of the binding agent contains structures that are thecomplement of the portion on the antigen to which the binding agentspecifically binds. The portion of the antigen to which an antibodybinds is called an “epitope.”

In certain embodiments, the binding agent is an antibody. Where thebinding agent that specifically binds a vimentin protein is an antibody,the antibody may be, without limitation, a polyclonal antibody, amonoclonal antibody, a chimeric antibody, a humanized antibody, agenetically engineered antibody, a bispecific antibody (where one of thespecificities of the bispecific antibody specifically binds to thevimentin protein), antibody fragments (including but not limited to“Fv,” “F(ab′)₂,” “F(ab),” and “Dab”); and single chains representing thereactive portion of an antibody (“SC-MAb”). Methods for makingantibodies and other binding agents are well known (see, e.g., Coliganet al., Current Protocols in Immunology, John Wiley and Sons, New YorkCity, N.Y., 1991; Jones et al., Nature 321: 522-525, 1986; Marx, Science229: 455-456, 1985; Rodwell, Nature 342: 99-100, 1989; Clackson, Br. J.Rheumatol. 3052: 36-39, 1991; Reichman et al., Nature 332: 323-327,1988; Verhoeyen, et al., Science 239: 1534-1536, 1988).

As used herein, “detectably labeled” means that a binding agent of theinvention is operably linked to a moiety that is detectable. “Operablylinked” means that the moiety is attached to the binding agent by eithera covalent or non-covalent (e.g., ionic) bond. Methods for creatingcovalent bonds are known (see general protocols in, e.g., Wong, S. S.,Chemistry of Protein Conjugation and Cross-Linking, CRC Press 1991;Burkhart et al., The Chemistry and Application of Amino CrosslinkingAgents or Aminoplasts, John Wiley & Sons Inc., New York City, N.Y.1999).

In accordance with the invention, a detectably labeled binding agent ofthe invention includes a binding agent that is conjugated to adetectable moiety. Another detectably labeled binding agent of theinvention is a fusion protein, where one partner is the binding agentand the other partner is a detectable label. Yet a further non-limitingexample of a detectably labeled binding agent is a first fusion proteincomprising a binding agent and a first moiety with high affinity to asecond moiety, and a second fusion protein comprising a second moietyand a detectable label. For example, a binding agent that specificallybinds to a vimentin protein may be operably linked to a streptavidinmoiety. A second fusion protein comprising a biotin moiety operablylinked to a fluorescein moiety may be added to the bindingagent-streptavidin fusion protein, where the combination of the secondfusion protein to the binding agent-streptavidin fusion protein resultsin a detectably labeled binding agent (i.e., a binding agent operablylinked to a detectable label).

According to the invention, a detectable label is a moiety that can betracked, and includes, without limitation, fluorophores (e.g.,fluorescein (FITC), phycoerythrin, rhodamine), chemical dyes, orcompounds that are radioactive, chemoluminescent, magnetic,paramagnetic, promagnetic, or enzymes that yield a product that may becolored, chemoluminescent, or magnetic. In particular embodiments, thedetectable label is detectable to a medical imaging device or system.For example, where the medical imaging system is an X-ray machine, thedetectable label that can be detected by the X-ray machine is aradioactive label (e.g., ³²P). Note that a binding agent need not bedirectly conjugated to the detectable moiety. For example, a bindingagent (e.g., a mouse anti-human vimentin antibody) that is itselfspecifically bound by a secondary detectable binding agent (e.g., a FITClabeled goat anti-mouse secondary antibody) is operably linked to adetectable moiety (i.e., the FITC moiety).

In some embodiments, measuring the level of expression of a vimentinprotein on the surface of the test damaged cell includes contacting theintact test damaged cell with a detectable binding agent thatspecifically binds to a vimentin protein. For example, where thedetectable binding agent is detectably labeled by being operably linkedto a fluorophore, cells staining with the fluorophore (i.e., those thatare specifically bound by the binding agent) can be identified byfluorescent activated cell sorter analysis (see Examples), or by routinefluorescent microscopy of clinical specimens prepared on slides.

In addition to detectable moieties, other non-limiting moieties that maybe operably linked to a binding agent of the invention include, withoutlimitation, a toxin (e.g., a radioactive isotope), an enzyme, anantibody (or a portion thereof), a cytotoxic drug, or a conjugate ofthese. Where a toxin is operably linked to a binding agent of theinvention, non-limiting examples of a toxin which can be operably linkedto a binding agent of the invention include a radioactive isotope,Diptheria toxin, a nuclease (e.g., DNAse or RNAse), a protease, adegradative enzyme, Pseudomonas exotoxin (PE), ricin A or B chains, andribonuclease A (Fizgerald D., Semin. Cancer Biol. 7: 87-95, 1996).

In some embodiments, the binding agent is an immunotoxin (e.g., anantibody-toxin conjugate or antibody-drug conjugate). Non-limitingexamples of immunotoxins include antibody-anthracycline conjugates(Braslawsky G. R. et al., European Patent No. EP0398305),antibody-cytokine conjugates (Gilles S. D., PCT Pub. No. WO9953958), andmonoclonal antibody-PE conjugates (Roffler S. R. et al., Cancer Res. 51:4001-4007, 1991).

In a further aspect, the invention provides a therapeutic compositioncomprising a cytotoxic drug, a binding agent that specifically binds toa vimentin protein or fragment thereof, and apharmaceutically-acceptable carrier. Non-limiting examples of suchpharmaceutically-acceptable carriers are described in more detail inRemington: The Science and Practice of Pharmacy, Gennaro et al. (eds),20^(th) Edition, Lippincott Williams & Wilkins, Philadelphia, Pa., 2001(ISBN 0-683-306472), a standard reference text. In certain embodiments,binding of the binding agent is toxic to damaged cells, regardless ofwhether such cells are drug-sensitive or multidrug resistant. In someembodiments, binding of the binding agent is toxic to neoplastic cells,regardless of whether such cells are drug-sensitive or multidrugresistant. In certain embodiments, the binding agent of the compositionis operably linked to a toxin.

Actual methods for preparing therapeutic compositions are known orapparent to those skilled in the art, and are described in detail inRemington: The Science and Practice of Pharmacy, 2001 (supra); and inPharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed., Williams& Wilkins (1995). The therapeutic compositions of the invention may bein any form suitable for administration including, without limitation,in the form of a tablet, a capsule, a powder, a solution, or an elixir.

Note that a cytotoxic drug of the therapeutic composition of theinvention need not be cytotoxic to all cells. In some embodiments, wherethe therapeutic composition is being administered to a patient sufferingfrom a disease caused by the presence of a damaged cell, the cytotoxicdrug of the therapeutic composition is an antipathogenic oranti-microbial drug. In some embodiments, where the damaged cells areinfected with a pathogen (e.g., a virus, a bacterium, or amulti-cellular parasite) and the disease is caused by the infection, thecytotoxic drug of the therapeutic composition is an antipathogenic drug.Where the damaged cells are infected by a pathogen, non-limitingexamples of the drug which differs depending upon the infecting pathogeninclude, but are not limited to, Acyclovir, amphotericin, ampicillin,anthracyclin, b-lactam antibiotics, cephalothin, chloramphenicol,chloroquine (CQ), cidofovir (CDV), ciprofloxacin, erythromycin,fluconazole, 5 flucytosine, fluoroquinolone, foscarnet, gancyclovir,halofantrine, Itraconazole, lamivudine, macrolides, mefloquine,methicillin, metronidazole, miconazole, nelfinavir, ofloxacin,penicillin, primaquine, quinoline, Streptomycin, Sulfonamides,teicoplanin, terbinafine, tetracycline, vancomycin, voriconazole.Therapeutically effective amounts of such drugs are known to routinelyskilled physicians and pharmacists. In addition, such information can beobtained from the manufacturer of the drug, or from the Physician's DeskReference, Medical Economics Co. (published yearly).

In some embodiments, where the therapeutic composition is beingadministered to a patient suffering from a cancer caused by the presenceof a neoplastic cell, the cytotoxic drug of the therapeutic compositionis an anti-cancer drug. Such anti-cancer drugs include, withoutlimitation, chemotherapeutic drugs and radiotherapeutic drugs.Non-limiting examples of such anti-cancer drugs include Actinomycin,Adriamycin (AR), Altretamine, Asparaginase, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin (DOX), Epoetin, Etoposide,Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,Ifosfamide, Imatinib, Irinotecan, Lomustine, Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin (MITO), Mitotane, Mitoxantrone,Paclitaxel, Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,Vinblastine (VLB), Vincristine, and Vinorelbine. Therapeuticallyeffective amounts of such drugs are known to routinely skilledphysicians and pharmacists. In addition, such information can beobtained from the manufacturer of the drug, or from the Physician's DeskReference, Medical Economics Co. (published yearly).

In another aspect, the invention provides a method for treating apatient suffering from a disease caused by the presence of damagedcells. This method includes administering to the patient atherapeutically effective amount of a drug and a therapeuticallyeffective amount of a binding agent that specifically binds to avimentin protein or fragment thereof. In some embodiments, the bindingagent kills damaged cells that are multidrug resistant while the drugkills damaged cells that are drug-sensitive. Of course, since thevimentin protein is expressed at moderate levels on drug-sensitivedamaged cells, the binding agent, which, in some embodiments isdifferent than the drug, will also kill drug-sensitive damaged cells.According to this method, the patient shows an improved prognosis forthe disease as compared to an untreated patient. In some embodiments,the untreated patient receives no binding agent, but does receive thedrug. An “untreated patient” or “control patient” is one that receivesno binding agent, but does receive the drug; or one that receives notreatment at all (i.e., receives neither the binding agent nor thedrug). The drug and the binding agent can be separately administered atdifferent times in any order or can be administered together. In someembodiments, the patient is a human.

In certain embodiments, the damaged cells of the patient are infectedwith a pathogen. In particular embodiments, the pathogen is a virus, abacterium, or a parasite (HIV, West Nile virus and Dengue virus;Mycobacteria, Rickettsia, and Chlamydia; Plasmodium, Leishmania, andTaxoplasma)

In another aspect, the invention provides a method for treating apatient suffering from a disease (e.g., cancer) caused by the presenceof neoplastic cells. This method includes administering to the patient atherapeutically effective amount of a drug and a therapeuticallyeffective amount of a binding agent that specifically binds to avimentin protein or fragment thereof. In some embodiments, the bindingagent kills neoplastic cells that are multidrug resistant while the drugkills neoplastic cells that are drug-sensitive. Since the vimentinprotein is expressed at moderate levels on drug-sensitive neoplasticcells, in some embodiments, the binding agent, which, in someembodiments is different than the drug, will also kill drug-sensitiveneoplastic cells. According to this method, the patient shows animproved prognosis for the disease as compared to an untreated patient.In some embodiments, the untreated patient receives no binding agent,but does receive the drug. In some embodiments, the untreated patientreceives no treatment at all (i.e., receives neither the binding agentnor the drug). The drug and the binding agent (e.g., an antibody) can beseparately administered in any order at different times or can beadministered together. In some embodiments, the patient is a human.

In certain embodiments, the neoplastic cells of the patient are breastcancer cells, ovarian cancer cells, myeloma cancer cells, lymphomacancer cells, melanoma cancer cells, sarcoma cancer cells, leukemiacancer cells, retinoblastoma cancer cells, hepatoma cancer cells, gliomacancer cells, mesothelioma cancer cells, or carcinoma cancer cells.

In some embodiments, the binding agent is operably linked to a toxin.Non-limiting examples of such toxins are described above.

As used herein, the term “therapeutically effective amount” is used todenote known treatments of a drug at dosages and for periods of timeeffective to kill a damaged cell. Administration may be by any routeincluding, without limitation, intravenous, parenteral, oral,sublingual, transdermal, topical, intranasal, intraocular, intravaginal,intrarectal, intraarterial, intramuscular, subcutaneous, andintraperitoneal.

The dose and dosage regimen of a binding agent, drug, and/or therapeuticcomposition in accordance with the invention, will depend mainly on thedegree of symptoms of the disease or cancer, the type of drug used(e.g., chemotherapeutic agent, radiotherapeutic agent, or antibiotic),the patient (e.g., the patient's gender, age, and/or weight), thepatient's history, and the patient's response to treatment. The doses ofbinding agent, drug, and/or therapeutic composition may be single dosesor multiple doses. If multiple doses are employed, the frequency ofadministration (schedule) will depend, for example, on the patient, typeof response, and type of drug used. Administration once a week may beeffective for some patients; whereas for others, daily administration oradministration every other day or every third day may be effective. Thepractitioner will be able to ascertain upon routine experimentation,which route of administration and frequency of administration are mosteffective in any particular case.

In yet another aspect, the invention features a method for detecting amultidrug resistant cell in a patient. The method includes administeringa binding agent that specifically binds to a vimentin protein orfragment thereof to a patient suspected of having a multidrug resistantcell, wherein the binding agent is operably linked to a label that isdetectable by a medical imaging device or system and examining thepatient with the medical imaging device or system. According to thismethod, the medical imaging device or system detects the binding agent(e.g., an antibody) specifically bound to the cell surface of amultidrug resistant cell in the patient.

Medical imaging devices and systems are known, as are labels that aredetectable by such systems. As discussed above, one non-limiting exampleof such a system and label is an X-ray machine which can detectradiolabeled binding agents. Other non-limiting examples of medicalimaging systems include (a) X-ray based Computer Tomography (CT),positron emission tomography (PET), and new combinations andimprovedments on these technologies [(PET+CT, spiral CT, single photonemission CT (SPECT), high resolution PET (microPET), andimmunoscintigraphy (using radiolabeled antibodies (Czernin, J. and M. E.Phelps, Ann. Rev. Med. 53:89-112, 2002; Goldenberg, D. M., Cancer 80(12):2431-2435, 1997; Langer, S. G. et al. (2001) World J. Surg.25:1428-1437; Middleton M L, Shell E G., Postgrad Med. 111(5):89-90,93-6, 2002); (b) magnetic resonance imaging (MRI) (Helbich, T. H., J.Radiol. 34:208-219, 2000; Langer, S. G. et al. World Journal of Surgery25:1428-1437, 2001; Nabi, H. A. and Zubeldia, J. M., Oncology J. NuclearMed. Technol. 30 (1):3-9, 2002); ultrasonic imaging (US) (Harvey, C. J.,et al. Advances in Ultrasound Clin. Radiol. 57:157-177, 2002; Langer, S.G. et al. W. J. Surg. 25:1428-1437, 2001); (c) fiber optic endoscope(Shelhase D E, Curr. Opin. Pediatr. 14:327-33, 2002); (d) gammascintillation detectors (detect gamma emitters, e.g. 192-Ir), and betascintillation detectors (detect beta emitters, e.g. 90-Sr/Y) (HanefeldC, Amirie, S. et al., Circulation 105:2493-6, 2002).

In certain embodiments, the patient is a mammal such as a human. Thepatient may be, for example, a human suffering from a disease caused bythe presence of the multidrug resistant cell. For example, the patientmay be suffering from cancer caused by a multidrug resistant neoplasticcell. Such a multidrug resistant neoplastic cell includes, withoutlimitation, an ovarian cancer cell, and myeloma cancer cell, a lymphomacancer cell, a melanoma cancer cell, a sarcoma cancer cell, a leukemiacancer cell, a retinoblastoma cancer cell a hepatoma cancer cell, aglioma cancer cell, a mesothelioma cancer cell, or a carcinoma cancercell.

In some embodiments, the multidrug resistant cell is a damaged cell, andthe patient is suffering from a disease caused by the presence of such amultidrug resistant damaged cell. Non-limiting ways in which a cell maybe damaged include infection by a pathogen (e.g., virus, bacteria orparasite), or a cell may suffer damage by necrosis. In particularembodiments, the damaged cell is infected with a pathogen (e.g., avirus, parasite, or bacterium). For example, the patient may besuffering from tuberculosis caused by a multidrug resistant strain ofMycobacterium tuberculosis.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds), Immunochemical Methods InCell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

4.2 Vimentin Antibodies

The invention provides antibodies directed against vimentin for use indetection, imaging and treatment of cancers and damaged (e.g.,pathogen-infected) cells. Anti-vimentin antibodies for use in theinvention are available from several commercial vendors. For example,CHEMICON (Temecula, Calif.) and ABR-Affinity BioReagents (Golden, Colo.)both produce such anti-human vimentin mouse monoclonal and/or rabbitpolyclonal antibodies.

The term “antibody” is used in the broadest sense and specificallycovers single anti-vimentin monoclonal and polyclonal antibodies, aswell as anti-vimentin antibody fragments (e.g., Fab, F(ab)2, and Fv) andanti-vimentin antibody compositions with polyepitopic specificity(including binding and non-binding antibodies). The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor-amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. Novel monoclonal antibodies or fragments thereof mean inprinciple all immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA ortheir subclasses such as the IgG subclasses or mixtures thereof. IgG andits subclasses are included, such as IgG1, IgG2, IgG2a, IgG2b, IgG3 orIgGM. The IgG subtypes IgG1/kappa and IgG 2b/kapp are also included asembodiments.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-vimentin antibody with a constant domain (e.g.,“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments (e.g., Fab,F(ab)2, and Fv), so long as they exhibit the desired biologicalactivity. (See, e.g., U.S. Pat. No. 4,816,567 and Mage & Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79-97(Marcel Dekker, Inc.), New York (1987)). Thus, the modified “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256:495 (1975), or may be madeby recombinant DNA methods (U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage libraries generated usingthe techniques described in McCafferty et al., Nature 348:552-554(1990), for example.

“Humanized” forms of non-human (e.g., murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)2 or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementary determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

Vimentin or anti-vimentin monoclonal antibodies or fragments thereofmean in principle all immunoglobulin classes such as IgM, IgG, IgD, IgE,IgA or their subclasses such as the IgG subclasses or mixtures thereof.IgG and its subclasses are, such as IgG1, IgG2, IgG2a, IgG2b, IgG3 orIgGM. The IgG subtypes IgG1/kappa and IgG 2b/kapp are included asembodiments. Fragments which may be mentioned are all truncated ormodified antibody fragments with one or two antigen-complementarybinding sites which show high binding and binding activity towardmammalian vimentin, such as parts of antibodies having a binding sitewhich corresponds to the antibody and is formed by light and heavychains, such as Fv, Fab or F(ab′)2 fragments, or single-strandedfragments. Truncated double-stranded fragments such as Fv, Fab orF(ab′)2 are. These fragments can be obtained, for example, by enzymaticmeans by eliminating the Fc part of the antibody with enzymes such aspapain or pepsin, by chemical oxidation or by genetic manipulation ofthe antibody genes. It is also possible and advantageous to usegenetically manipulated, non-truncated fragments. The anti-vimentinantibodies or fragments thereof can be used alone or in mixtures.

The novel antibodies, antibody fragments, mixtures or derivativesthereof advantageously have a binding affinity for vimentin with adissociation constant value within a log-range of from about 1×10⁻¹¹ M(0.01 nM) to about 1×10⁻⁸ M (10 nM), or about 1×10⁻¹⁰ M (0.1 nM) toabout 3×10⁹⁹ M (3 nM).

The antibody genes for the genetic manipulations can be isolated, forexample from hybridoma cells, in a manner known to the skilled worker.For this purpose, antibody-producing cells are cultured and, when theoptical density of the cells is sufficient, the mRNA is isolated fromthe cells in a known manner by lysing the cells with guanidiniumthiocyanate, acidifying with sodium acetate, extracting with phenol,chloroform/isoamyl alcohol, precipitating with isopropanol and washingwith ethanol. cDNA is then synthesized from the mRNA using reversetranscriptase. The synthesized cDNA can be inserted, directly or aftergenetic manipulation, for example by site-directed mutagenesis,introduction of insertions, inversions, deletions or base exchanges,into suitable animal, fungal, bacterial or viral vectors and beexpressed in appropriate host organisms. Preference is given tobacterial or yeast vectors such as pBR322, pUC18/19, pACYC184, lambda oryeast mu vectors for the cloning of the genes and expression in bacteriasuch as E. coli or in yeasts such as Saccharomyces cerevisiae.

The invention furthermore relates to cells that synthesize vimentinantibodies. These include animal, fungal, bacterial cells or yeast cellsafter transformation as mentioned above. They are advantageouslyhybridoma cells or trioma cells, preferably hybridoma cells. Thesehybridoma cells can be produced, for example, in a known manner fromanimals immunized with vimentin and isolation of theirantibody-producing B cells, selecting these cells for vimentin-bindingantibodies and subsequently fusing these cells to, for example, human oranimal, for example, mouse mylemoa cells, human lymphoblastoid cells orheterohybridoma cells (see, e.g., Koehler et al., (1975) Nature 256:496) or by infecting these cells with appropriate viruses to produceimmortalized cell lines. Hybridoma cell lines produced by fusion areparticularly useful, mouse hybridoma cell lines are very useful. Thehybridoma cell lines of the invention secrete antibodies of the IgGtype. The binding of the mAb antibodies of the invention bind with highaffinity to vimentin.

The invention further includes derivates of these anti-vimentin, whichpreferably retain their vimentin-binding activity while altering one ormore other properties related to their use as a pharmaceutical agent,e.g., serum stability or efficiency of production. Examples of suchantivimentin antibody derivatives include peptides, peptidomimeticsderived from the antigen-binding regions of the antibodies, andantibodies, fragments or peptides bound to solid or liquid carriers suchas polyethylene glycol, glass, synthetic polymers such aspolyacrylamide, polystyrene, polypropylene, polyethylene or naturalpolymers such as cellulose, Sepharose or agarose, or conjugates withenzymes, toxins or radioactive or nonradioactive markers such as ³H,¹²³I, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁵Fe, ⁵⁹Fe, ⁹⁰Y, ^(99m)Tc(metastable isomer of Technetium 99), ⁷⁵Se, or antibodies, fragments orpeptides covalently bonded to fluorescent/chemiluminescent labels suchas rhodamine, fluorescein, isothiocyanate, phycoerythrin, phycocyanin,fluorescamine, metal chelates, avidin, streptavidin or biotin.

The novel antibodies, antibody fragments, mixtures and derivativesthereof can be used directly, after drying, for example freeze drying,after attachment to the abovementioned carriers or after formulationwith other pharmaceutical active and ancillary substances for producingpharmaceutical preparations. Examples of active and ancillary substanceswhich may be mentioned are other antibodies, antimicrobial activesubstances with a microbiocidal or microbiostatic action such asantibiotics in general or sulfonamides, antitumor agents, water,buffers, salines, alcohols, fats, waxes, inert vehicles or othersubstances customary for parenteral products, such as amino acids,thickeners or sugars. These pharmaceutical preparations are used tocontrol diseases, preferably to control arthritic disturbances,advantageously disturbances of joint cartilage.

The anti-vimentin antibodies of the invention can be administered orallyor parenterally subcutaneously, intramuscularly, intravenously orinterperitoneally.

The antibodies, antibody fragments, mixtures or derivatives thereof canbe used in therapy or diagnosis directly or after coupling to solid orliquid carriers, enzymes, toxins, radioactive or nonradioactive labelsor to fluorescent/chemiluminescent labels as described above. Vimentincan be detected on a wide variety of cell types—particularly neoplasticcells. The human vimentin monoclonal antibody of the present inventionmay be obtained as follows. Those of skill in the art will recognizethat other equivalent procedures for obtaining vimentin antibodies arealso available and are included in the invention.

First, a mammal is immunized with human vimentin. Purified humanvimentin is commercially available from Sigma (St. Louis, Mo., catalogA6152), as well as other commercial vendors. Human vimentin may bereadily purified from human placental tissue. Furthermore, methods ofimmunoaffinity purification for obtaining highly purified vimentinimmunogen are known (see, e.g., Vladutiu et al., (1975) 5: 147-59 Prep.Biochem.). The mammal used for raising anti-human vimentin antibody isnot restricted and may be a primate, a rodent such as mouse, rat orrabbit, bovine, sheep, goat or dog.

Next, antibody-producing cells such as spleen cells are removed from theimmunized animal and are fused with myeloma cells. The myeloma cells arewell-known in the art (e.g., p3x63-Ag8-653, NS-0, NS-1 or P3U1 cells maybe used). The cell fusion operation may be carried out by a well-knownconventional method.

The cells, after being subjected to the cell fusion operation, are thencultured in HAT selection medium so as to select hybridomas. Hybridomas,which produce antihuman monoclonal antibodies, are then screened. Thisscreening may be carried out by, for example, sandwich ELISA(enzyme-linked immunosorbent assay) or the like in which the producedmonoclonal antibodies are bound to the wells to which human vimentin isimmobilized. In this case, as the secondary antibody, an antibodyspecific to the immunoglobulin of the immunized animal, which is labeledwith an enzyme such as peroxidase, alkaline phosphatase, glucoseoxidase, beta-D-galactosidase or the like, may be employed. The labelmay be detected by reacting the labeling enzyme with its substrate andmeasuring the generated color. As the substrate, 3,3-diaminobenzidine,2,2-diaminobis-o-dianisidine, 4-chloronaphthol, 4-aminoantipyrine,o-phenylenediamine or the like may be produced.

By the above-described operation, hybridomas, which produce anti-humanvimentin antibodies, can be selected. The selected hybridomas are thencloned by the conventional limiting dilution method or soft agar method.If desired, the cloned hybridomas may be cultured on a large scale usinga serum-containing or a serum free medium, or may be inoculated into theabdominal cavity of mice and recovered from ascites, thereby a largenumber of the cloned hybridomas may be obtained.

From among the selected anti-human vimentin monoclonal antibodies, thosethat have an ability to bind cell surface vimentin are then chosen forfurther analysis and manipulation.

The monoclonal antibodies herein further include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-vimentin antibody with a constant domain (e.g.,“humanized” antibodies), or a light chain with a heavy chain, or a chainfrom one species with a chain from another species, or fusions withheterologous proteins, regardless of species of origin or immunoglobulinclass or subclass designation, as well as antibody fragments (e.g., Fab,F(ab)2, and Fv), so long as they exhibit the desired biologicalactivity. (See, e.g., U.S. Pat. No. 4,816,567 and Mage & Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79-97(Marcel Dekker, Inc.), New York (1987)).

Thus, the term “monoclonal” indicates that the character of the antibodyobtained is from a substantially homogeneous population of antibodies,and is not to be construed as requiring production of the antibody byany particular method. For example, the monoclonal antibodies to be usedin accordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256:495 (1975), ormay be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage librariesgenerated using the techniques described in McCafferty et al., Nature348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g., murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)2 or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementary determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody comprises substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also comprises atleast a portion of an immununoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source, which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., (1986) Nature 321: 522-525; Riechmann et al., (1988)Nature, 332: 323-327; and Verhoeyen et al., (1988) Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies, wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., (1993) J.Immunol., 151:2296; and Chothia and Lesk (1987) J. Mol. Biol., 196:901).Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., (1992) Proc. Natl. Acad. Sci.(USA), 89: 4285; and Presta et al., (1993) J. Immunol., 151:2623).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding.

Human antibodies directed against vimentin are also included in theinvention. Such antibodies can be made, for example, by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor (1984) J. Immunol., 133, 3001; Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., (1991) J.Immunol., 147:86-95. Specific methods for the generation of such humanantibodies using, for example, phage display, transgenic mousetechnologies and/or in vitro display technologies, such as ribosomedisplay or covalent display, have been described (see Osbourn et al.(2003) Drug Discov. Today 8: 845-51; Maynard and Georgiou (2000) Ann.Rev. Biomed. Eng. 2: 339-76; and U.S. Pat. Nos. 4,833,077; 5,811,524;5,958,765; 6,413,771; and 6,537,809.

It is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch gem-line mutant mice will result in the production of humanantibodies upon antigen challenge (see, e.g., Jakobovits et al., (1993)Proc. Natl. Acad. Sci. (USA), 90: 2551; Jakobovits et al., (1993)Nature, 362:255-258; and Bruggermann et al., (1993) Year in Immuno.,7:33).

Alternatively, phage display technology (McCafferty et al., (1990)Nature, 348: 552-553) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats (for review see, e.g., Johnson et al., (1993) Curr.Opin. in Struct. Bio., 3:564-571). Several sources of V-gene segmentscan be used for phage display. For example, Clackson et al., ((1991)Nature, 352: 624-628) isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Marks et al., ((1991) J. Mol.Biol., 222:581-597, or Griffith et al., (1993) EMBO J., 12:725-734).

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling” (see Marks et al.,(1992) Bio/Technol., 10:779-783). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thenM range. A strategy for making very large phage antibody repertoireshas been described by Waterhouse et al., ((1993) Nucl. Acids Res.,21:2265-2266).

Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e., the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT WO 93/06213,published 1 Apr. 1993). Unlike traditional humanization of rodentantibodies by CDR grafting, this technique provides completely humanantibodies, which have no framework or CDR residues of rodent origin.

By using the above-described monoclonal antibody of the presentinvention, human vimentin in a sample can be detected or quantified. Thedetection or quantification of the human vimentin in a sample can becarried out by an immunoassay utilizing the specific binding reactionbetween the monoclonal antibody of the present invention and humanvimentin. Various immunoassays are well-known in the art and any of themcan be employed. Examples of the immunoassays include sandwich methodemploying the monoclonal antibody and another monoclonal antibody asprimary and secondary antibodies, respectively, sandwich methodsemploying the monoclonal antibody and a polyclonal antibody as primaryand secondary antibodies, staining methods employing gold colloid,agglutination method, latex method and chemical luminescence. Amongthese, especially is sandwich ELISA. As is well-known, in this method, aprimary antibody is immobilized on, for example, the inner wall of awell and then a sample is reacted with the immobilized primary antibody.After washing, a secondary antibody is reacted with the antigen-antibodycomplex immobilized in the well. After washing, the immobilizedsecondary antibody is quantified. As the primary antibody, an antibodyspecifically reacts with human vimentin is preferably employed.

The quantification of the secondary antibody may be carried out byreacting a labeled antibody (e.g., enzyme-labeled antibody) specific tothe immunoglobulin of the animal from which the secondary antibody wasobtained with the secondary antibody, and then measuring the label.Alternatively, a labeled (e.g., enzyme-labeled) antibody is used as thesecondary antibody and the quantification of the secondary antibody maybe carried out by measuring the label on the secondary antibody.

4.3 Vimentin Binding Agents

In another aspect, the invention provides a binding agent thatspecifically binds to a vimentin protein or fragment thereof. As usedherein, “specifically binds” means that a binding agent recognizes andbinds to a vimentin protein or fragment thereof, but does notsubstantially recognize and bind to other molecules in a sample. Thus, abinding agent of the invention specifically binds to the surface of aMDR cell that expresses vimentin on its cell surface. A useful bindingagent forms an association with the vimentin protein with an affinity ofat least at least about 10⁶M-1, or at least about 10⁷ M-1, or at leastabout 10⁸ M-1, or at least about 10⁹ M-1 either in water, underphysiological conditions, or under conditions which approximatephysiological conditions with respect to ionic strength, e.g., 140 mMNaCl, 5 mM MgCl₂. As used herein, a “binding agent” is a molecule thatspecifically binds or attaches to a vimentin protein or fragmentthereof.

A binding agent need not be any particular size or have any particularstructure so long as it specifically binds to the vimentin protein orfragment thereof. Thus, a “binding agent” is a molecule thatspecifically binds to or attaches to any region (e.g., three dimensionalstructure, amino acid sequence, or particular small chemical groups) solong as it specifically binds to the vimentin protein or fragmentthereof. Non-limiting examples of binding agents include natural ligands(such as hormones or GTP), as well as synthetic small molecules,chemicals, nucleic acids, peptides, and proteins such as hormones,antibodies, and portions thereof.

There are a number of examples of non-antibody vimentin binding agentsknown in the art. For example, modified LDL has been shown to bindspecifically to vimentin at an affinity of approximately Kd of 1.7×10⁻⁷M (corresponding to a Ka of about 5.9×10⁶ M-1) (see Heidenthal, et al.(2000) Biochem. Biophys. Res. Comm. 267: 49-53). The modified LDL is,optimally, oxidized and acetylated LDL. Methods of producing suchmodified LDL are known in the art. For example, oxidized LDL may beprepared by incubating LDL with 5 uM CuSO₄ in EDTA-free, O₂-saturatedPBS (see Hrboticky et al. (1999) Arterioscler. Throm. Basc. Biol. 19:1267-75). Acetylation, and iodination, of the oxidized LDL may beperformed using methods known in the art (see Basu et al. (1976) Proc.Natl. Acad. Sci. USA 73: 3178-82 (acetylation) and Bilheimer et al.(Biochim. Biophys. Acta 260: 212-21 (iodination)). Other examples ofnon-antibody vimentin binding agents include the Nlk1 protein (U.S. Pat.No. 6,476,193) and SNAP23 protein (see Faigle et al. (2000) Mol. Biol.Cell. 11: 3485-3494), as well as desmin, glial fibrillary acidicprotein, and peripherin, fimbrin, RhoA-binding kinase alpha and proteinphosphatase 2A. Vimentin itself is known to multimerize throughself-association, and so, is also a non-antibody vimentin binding agent.

4.4 Vimentin-Targeted Diagnostics

The invention further allows the early identification of patients havingsuch MDR neoplastic or damaged cells. For example, where the patientidentified as having such cells is a patient in remission of cancer oris being treated for cancer (e.g., a patient suffering from breastcancer, ovarian cancer, prostate cancer, leukemia, etc.), the inventionallows identification of these patients prior to resurgence and/orprogression of their cancer, as well as allows the monitoring of thesepatients during treatment with a drug, such that the treatment regimencan be altered. Similarly, where the patient identified as having suchcells is an asymptomatic patient who is being treated for an infectiousdisease, or had received treatment for an infectious disease (e.g.,hepatitis B), the invention allows identification of these patientsprior to resurgence of symptoms, as well as allows the monitoring ofthese patients during treatment with a drug, such that the treatmentregimen can be altered if such MDR cells are detected. Furthermore,diagnostic applications of the invention allow early diagnosis andimaging of neoplastic, MDR neoplastic or damaged (e.g.,pathogen-infected) cells using the cell surface vimentin marker

The diagnostic applications of the invention include probes and otherdetectable agents that are joined to a vimentin binding agent, such asan anti-vimentin antibody. As used herein, the term “detectably labeled”means that a binding agent of the invention is operably linked to amoiety that is detectable. “Operably linked” means that the moiety isattached to the binding agent by either a covalent or non-covalent(e.g., ionic) bond. Methods for creating covalent bonds are known (seegeneral protocols in, e.g., Wong, S. S., Chemistry of ProteinConjugation and Cross-Linking, CRC Press 1991; Burkhart et al., TheChemistry and Application of Amino Crosslinking Agents or Aminoplasts,John Wiley & Sons Inc., New York City, N.Y. 1999).

In accordance with the invention, a detectably labeled binding agent ofthe invention includes a binding agent that is conjugated to adetectable moiety. Another detectably labeled binding agent of theinvention is a fusion protein, where one partner is the binding agentand the other partner is a detectable label. Yet a further non-limitingexample of a detectably labeled binding agent is a first fusion proteincomprising a binding agent and a first moiety with high affinity to asecond moiety, and a second fusion protein comprising a second moietyand a detectable label. For example, a binding agent that specificallybinds to a vimentin protein may be operably linked to a streptavidinmoiety. A second fusion protein comprising a biotin moiety operablylinked to a fluorescein moiety may be added to the bindingagent-streptavidin fusion protein, where the combination of the secondfusion protein to the binding agent-streptavidin fusion protein resultsin a detectably labeled binding agent (i.e., a binding agent operablylinked to a detectable label).

The detectable label of the invention is a moiety that can be tracked,and includes, without limitation, fluorophores (e.g., fluorescein(FITC), phycoerythrin, rhodamine), chemical dyes, or compounds that areradioactive, chemoluminescent, magnetic, paramagnetic, promagnetic, orenzymes that yield a product that may be colored, chemoluminescent, ormagnetic. In particular embodiments, the detectable label is detectableto a medical imaging device or system. For example, where the medicalimaging system is an X-ray machine, the detectable label that can bedetected by the X-ray machine is a radioactive label (e.g., ³²P). Notethat a binding agent need not be directly conjugated to the detectablemoiety. For example, a binding agent (e.g., a mouse anti-human vimentinantibody) that is itself specifically bound by a secondary detectablebinding agent (e.g., a FITC labeled goat anti-mouse secondary antibody)is operably linked to a detectable moiety (i.e., the FITC moiety).

In some embodiments, measuring the level of expression of a vimentinprotein on the surface of the test damaged cell includes contacting theintact test damaged cell with a detectable binding agent thatspecifically binds to a vimentin protein. For example, where thedetectable binding agent is detectably labeled by being operably linkedto a fluorophore, cells staining with the fluorophore (i.e., those thatare specifically bound by the binding agent) can be identified byfluorescent activated cell sorter analysis (see Examples), or by routinefluorescent microscopy of clinical specimens prepared on slides.

Medical imaging devices and systems are known, as are labels that aredetectable by such systems. As discussed above, one non-limiting exampleof such a system and label is an X-ray machine which can detectradiolabeled binding agents. Other non-limiting examples of medicalimaging systems include (a) X-ray based Computer Tomography (CT),positron emission tomography (PET), and new combinations andimprovedments on these technologies [(PET+CT, spiral CT, single photonemission CT (SPECT), high resolution PET (microPET), andimmunoscintigraphy (using radiolabeled antibodies (Czemin, J. and M. E.Phelps (2002) Annual Reviews of Medicine 53:89-112; Goldenberg, D. M.(1997) Cancer 80 (12):2431-2435; Langer, S. G. et al. (2001) WorldJournal of Surgery 25:1428-1437; Middleton M L and Shell E G (2002)Postgrad Med 111(5):89-90, 93-6; (b) magnetic resonance imaging (MRI)(Helbich, T. H, (2002) Journal of Radiology 34:208-219; Langer, S. G. etal. (2001) World Journal of Surgery 25:1428-1437; Nabi, H. A. andZubeldia, J. M. (2002) Oncology Journal of Nuclear Medicine Technology30 (1):3-9); ultrasonic imaging (US) (Harvey, C. J, et al. (2002)Advances in Ultrasound Clinical Radiology 57:157-177; Langer, S. G. etal. (2001) World Journal of Surgery 25:1428-1437); (c) fiber opticendoscope (Shelhase D E (2002) Curr Opin Pediatr 14:327-33); (d) gammascintillation detectors (detect gamma emitters, e.g. 192-Ir), and betascintillation detectors (detect beta emitters, e.g. 90-Sr/Y) (HanefeldC, Amirie, S. et al. (2002) Circulation 105:2493-6, 2002).

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to vimentin polypeptide can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or disordersassociated with the aberrant expression of a cell surface vimentin. Theinvention provides for the detection of aberrant expression of cellsurface vimentin (a) assaying the expression of the polypeptide ofinterest in cells or cell surface membrane fractions of an individualusing one or more antibodies specific to vimentin and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed cell surface vimentin expressionlevel compared to the standard expression level is indicative ofaberrant expression. For example, where multidrug resistance in aneoplastic cell is to be detected, the “standard expression level” towhich comparison should be made is a nonmultidrug resistant neoplasticcell of the same or similar origin or cell type. Similarly, whereneoplasia in a test cell is to be detected, the “standard expressionlevel” to which comparison should be made is a non-neoplastic cell ofthe same or similar origin or cell type. Furthermore, where “damage” ina test cell (e.g., pathogen infection) is to be detected, the “standardexpression level” to which comparison should be made is a non-damagecell (e.g., uninfected cell) of the same or similar origin or cell type.

With respect to cancer, the presence of a relatively high amount of cellsurface vimentin in biopsied tissue or test cell from an individual mayindicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, M., et al., (1985) J.Cell. Biol. 101:976-985); Jalkanen, M., et al. (1987) J. Cell. Biol.105:3087-3096). Other antibody-based methods useful for detectingvimentin protein expression include immunoassays, such as the enzymelinked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In) andtechnetium (⁹⁹Tc); luminescent labels, such as luminol; and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of cell surface vimentinin an animal, such as a mammal, e.g., a human. In one embodiment,diagnosis comprises: a) administering (for example, parenterally,subcutaneously, or intraperitoneally) to a subject an effective amountof a labeled anti-vimentin antibody, or other vimentin binding agent,which specifically binds to cell surface vimentin in the animal; b)waiting for a time interval following administration for permitting thelabeled molecule to preferentially concentrate at sites in the subjectwhere the polypeptide is expressed (and for unbound labeled molecule tobe cleared to background level); c) determining background level; and d)detecting the labeled molecule in the subject, such that detection oflabeled molecule above the background level indicates that the subjecthas a particular disease or disorder associated with aberrant expressionof the polypeptide of interest. Background level can be determined byvarious methods including, comparing the amount of labeled moleculedetected to a standard value previously determined for a particularsystem.

A vimentin-specific antibody or antibody portion which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, ¹³¹I, ¹¹¹In, ^(99m)Tc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously or intraperitoneally) into themammal to be examined for a disorder. Generally, suitable radioisotopesfor imaging and detection include radioisotopes that emit alpha, beta,or gamma radiation. Gamma radiation may be particularly easy to imageusing current technology. Examples are radioisotopes derived fromGallium, Indium, Technetium, Yttrium, Ytterbium, Rhenium, Platinum,Thallium, and Astatine, e.g., ⁶⁷Ga, ¹¹¹In, ^(99m)Tc, ⁹⁰Y, ⁸⁶Y, ¹⁶⁹Yb,¹⁸⁸Re, ^(195m)Pt, ²⁰¹Ti, and ²¹¹At. It is understood in the art that thesize of the subject and the imaging system used will determine thequantity of imaging moiety needed to produce diagnostic images. In thecase of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ^(99m)Tc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which expresscell surface vimentin protein. Reagents and methods for tumor imaging invivo (i.e., in situ) are known in the art and described in, for example,S. W. Burchiel et al. (1982) “Immunopharmacokinetics of RadiolabeledAntibodies and Their Fragments.” (Chapter 13 in Tumor Imaging. TheRadiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,eds., Masson Publishing Inc.). For example, antibody labels or markersfor in vivo imaging of endokine alpha protein include those detectableby X-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

Depending on several variables which can be optimized using routinepractice, including the type of label used and the mode ofadministration, the time interval following the administration forpermitting the labeled molecule to preferentially concentrate at sitesin the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In certain embodiment, monitoring of the disease or disorder is carriedout by repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Significantly, the presence of the labeled anti-vimentin antibody orother vimentin binding can be detected in the patient using methodsknown in the art for in vivo scanning. These methods depend upon thetype of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

For example, in a specific embodiment, the anti-vimentin antibody islabeled with a radioisotope and is detected in the patient using aradiation responsive surgical instrument (Thurston et al., U.S. Pat. No.5,441,050). In another embodiment, the anti-vimentin antibody is labeledwith a fluorescent compound and is detected in the patient using afluorescence responsive scanning instrument. In another embodiment, theanti-vimentin antibody is labeled with a positron emitting metal and isdetected in the patent using positron emission-tomography. In yetanother embodiment, the anti-vimentin antibody is labeled with aparamagnetic label and is detected in a patient using magnetic resonanceimaging (MRI).

4.5 Vimentin-Targeted Therapeutics

The invention takes advantage of the fact that vimentin protein cellsurface marker is present only in negligible levels on the surface ofnormal cells of the body, but occurs on the cell surface of neoplasticand, expecially, in multidrug resistant neoplastic cells. In contrast,other markers, and particularly the MDR markers such as P-glycoproteinand the multidrug resistance protein (MRP), are present at variablelevels on the surface of many different normal cell and tissues,including high levels on the surface of liver, kidney, stem cells, andblood-brain barrier epithelial cells. Accordingly, the inventionprovides a highly specific way of targeting therapeutics to neoplasticand, particularly, multidrug resistant neoplastic, cells using a bindingagent that binds to cell surface vimentin.

Therapeutic agents to be targeted to vimentin by the methods of theinvention include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Anti-Vimentin Antibodies

In one approach, anti-vimentin antibodies specific to cell surfaceexpressed vimentin expressed on damaged (e.g., pathogen-infected),neoplastic, and MDR neoplastic cells are administered systemically to apatient with cancer. Adhesion of antibody to tumor cells results intumor cell death by activation of the complement system(complement-mediated cytotoxicity) or by activation of T cells(antibody-dependent cell-mediated cytotoxicity). Other antibody-inducedantitumor effects include induction of apoptosis, enhancement of thecytotoxic effects of a second agent (e.g., an anti-cancerchemotherapeutic drug), and induction of anti-idiotype network response.In certain embodiments, humanized anti-vimentin antibodies may beutilized. Humanized antibodies avoid the potential problem of causinghuman patients to develop anti-animal (e.g., anti-mouse or anti-rat)antibodies. Humanized antibodies consist of human antibody containt thecompelementarity-determining region from a nonhuman source.

Antibody based therapeutics have been used successfully in a number ofcases. For example, Rituximab is a genetically engineered monoclonalanti-CD20 antibody used to treat non-Hodgkins lymphoma (NHL), arelatively common malignancy affecting both young and old populations.The CD20 antigen typically present on these B-cell lymphomas serves asan ideal targeting antigen because it is not present on plasma cells,B-cell precursors, stem cells, or dendritic (antigen-presenting) cells.The Rituximab antibody (or Rituxan) is neither shed nor internalized byNHL cells and it does not undergo modification following antigenbinding. Rituximab was approved by the FDA in 1997 for the treatment ofrelapsed or refractory CD20-positive B-cell NHL and for low-grade orfollicular type lymphoma (see Abou-Jawde et al. (2003) Clin. Therap. 25:2121-37; and Kim (2003) Am. J. Surg. 186: 264-68). It functions bymediating antibody-dependent cytotoxicity, inhibiting cell growth,sensitizing chemoresistant cells to toxins and chemotherapy, andinducing apoptosis in a dose-dependent manner (White et al. (2001) Annu.Rev. Med. 52: 125-45; Press (1999) Semin. Oncol. 26 (Suppl 14): 58-65).

Another example of an anti-tumor antigen antibody therapeutic isAlemtuzumab. Alemtuzumab is a humanized anti-CD52 monoclonal antibodyapproved by the FDA in 2001 for the treatment of patients with B-cellchronic lymphocytic leukemia (CLL), a prevalent form of adult malignancy(see Abou-Jawde et al. (2003) Clin. Therap. 25: 2121-37). Even thoughthe function of CD52 is not well identified, Alemtuzumab has been shownto elicit tumor response even in the presence of bulky disease(Ferrajoli et al. (2001) Expert Opin. Biol. Ther. 1: 1059-1065).

Still another example of an anti-tumor antigen antibody therapeutic isTrastuzumab (also Herceptin), a recombinant humanized anti-HER 2monoclonal antibody approved by the FDA in 1998 for the treatment ofmetastatic breast cancer (see Abou-Jawde et al. (2003) Clin. Therap. 25:2121-37; and Kim (2003) Am. J. Surg. 186: 264-68). HER 2 is an epidermalgrowth factor receptor (EGFR) family member expressed by many breastcancers tumors and, accordingly, this antibody therapeutic is effectiveagainst solid tumors. Several randomized, controlled studies wereconducted and showed efficacy and improved quality of life inHER2-positive breast cancer patients treated with Trastuzumab (Vogel etal. (2002) J. Clin. Oncol. 20: 719-26).

Finally, Cetuximab is a chimeric anti-HER1 monoclonal antibody that iseffective in treating several HER1/erb-B1 expressing solid tumors,including colorectal, pancreatic, non-small cell lung cancer (NSCLC),and head and neck cancers (see, e.g., O'dwyer et al. (2002) Semin.Oncol. 29 (Suppl. 14): 10-17). Cetuximab competes for the binding of theEGFR and removes receptor from the cell membrane by stimulatinginternalization and thereby dirsuptin the cellular process responsiblefor proliferation growth and metastasis (see Abou-Jawde et al. (2003)Clin. Therap. 25: 2121-37).

Vimentin-Targeted Antibody and Ligand Conjugates

In addition to the ‘naked’ antibody approach described above, antibodiescan be conjugated, or otherwise “operably linked” to biological orchemical toxins or radioisotopes. An anti-vimentin antibody or antibodyfragment thereof may be conjugated or otherwise operably linked to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. “Operably linked”means that the therapeutic moiety is attached to the binding agent byeither a covalent or non-covalent (e.g., hydrophobic or ionic) bond.Methods for creating covalent bonds are known (see general protocols in,e.g., Wong, S. S., Chemistry of Protein Conjugation and Cross-Linking,CRC Press 1991; Burkhart et al., The Chemistry and Application of AminoCrosslinking Agents or Aminoplasts, John Wiley & Sons Inc., New YorkCity, N.Y. 1999). Following systemic administration, the therapy istargeted to the cancer cell (or MDR cancer cell or damaged (e.g.,pathogen-infected) cell) by the antibody.

The invention further includes vimentin-targeted agents made up of avimentin targeting element and a toxic agent, such as a biologicaltoxin, a chemical toxin or a radioisotope. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Biological toxins have been conjugated, orgenetically fused in frame, to antibodies, and other tumormarker-localizing agents. These biological toxins include ricin,diphtheria toxin and Pseudomonas exotoxin. Following binding to cellsurface vimentin, the toxins generally cross the cell membrane, and maythen be processed, before killing the targeted cell. The toxic effect istypically due to inhibition of protein-synthesis by the activebiological toxin.

For example, in one embodiment, anti-vimentin antibodies are conjugatedto cobra venom factor. In accordance with the invention, vimentinspecific antibodies conjugated to cobra venom factor are used to treatcancer, including especially multidrug resistant cancer in a human.Methods of conjugating antibodies to cobra venom factor are taught inU.S. Pat. No. 5,773,243. In some embodiments, the binding agent is animmunotoxin (e.g., an antibody-toxin conjugate or antibody-drugconjugate). Non-limiting examples of immunotoxins includeantibody-anthracycline conjugates (Braslawsky G. R. et al., EuropeanPatent No. EP0398305), antibody-cytokine conjugates (Gilles S. D., PCTPublication No. WO9953958), and monoclonal antibody-PE conjugates(Roffler S. R. et al., Cancer Res. 51: 4001-4007, 1991).

Techniques for conjugating other therapeutic moieties to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982); each of which is incorporated herein byreference. Alternatively, an antibody can be conjugated to a secondantibody to form an antibody heteroconjugate as described by Segal inU.S. Pat. No. 4,676,980, which is incorporated herein by reference.

Drug Attachment

A number of approaches to drug and therapeutics attachment and releasehave been described in the literature, and the strategies employed insoluble polymer-drug conjugates have been recently reviewed (Soyez, etal., (1966) Adv. Drug Del. Rev. 21:81-106). The chemistry of many ofthese conjugation methods is described in textbooks (e.g., Ref. Wong(1991) CRC Press, Boca Raton, Fla.), site of the attachment to theantibody.

The most used site is that of the e-amino groups of the lysine residues,as these are chemically convenient to use, either by amide bond formingreagents such as carbodiimides or by heterobifunctional agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson, et al.,(1978) Biochem J. 173:723-737) which can introduce reactive thiolgroups. Antibodies have a variable number of lysine residues, which arespread over the whole of the antibody, and there is no evidence for anysubset of more reactive residues. Consequently, lysine residues at theactive site are as likely to be modified as any other residues leadingto loss of antibody activity, and the greater the number of lysineresidues modified, the greater the likelihood of loss of binding.Linking to lysine ε-amino groups can also have other effects. Firstly byneutralizing a positive charge on the protein could have structuraleffects, or affect the solubility of the antibody (Hudecz, et al.,(1990) Bioconjug. Chem. 1:197-204) although this can be overcome byusing a reagent such as 2-iminothiolane which provides a thiolfunctionality without altering the charge (Jue, et al., (1978)Biochemistry 17:5399-5406). Finally, the number of lysine residuesmodified is statistical, so a range of modified residues is providedwithin a population of antibody molecules, leading to a variable amountof drug attached and loss of binding activity within a singlepreparation (Firestone, et al., (1996) BR96-Dox, J. Control 39:251-259).

A second site for modification is the sugar residues attached to thehinge region of the antibody. As this is a site of unique chemicalreactivity situated away from the antibody binding site this is a usefularea for attachment. This has been exploited by several groups byperiodate oxidation of the sugars to provide aldehyde groups(O'Shanessy, et al., (1984) Immunol. Lett. 8:273-277; O'Shanessy, etal., (1987) J. Immunol. Methods 99:153-161; and Rodwell, et al., (1986)Proc. Natl. Acad. Sci. USA 83:2632-2636) which can be used in a numberof coupling procedures. Aldehyde groups can also be generated onimmunoglobulins by an enzymic reaction involving neuraminidase andglucose oxidase (Rodwell, et al., (1986) Proc. Natl. Acad. Sci. USA83:2632-2636 and Stan, et al., (1999) Cancer Res. 59:115-121).Antibodies have also been modified by genetic engineering to produce newoligosaccharides sites which are reported to be more favourably locatedfor attachment of carrier-drug molecules (Qu, et al., (1998) J. Immunol.Methods 213:131-144).

The third major possibility for attachment is through internaldisulphide bonds within the antibody. Disulphide linkages play animportant role in the structure of antibodies, providing both interchainand intrachain linkages. The intrachain linkages which stabilize theantibody domain structure can be selectively cleaved by dithiothreitolwithout affecting the interchain disulphide holding the antibody chainstogether. This procedure has been exploited by (Willner et al., (1993)Bioconjug. Chem. 4:521-527) to produce more soluble conjugates withbetter binding activity and with a defined number of drug molecules perantibody molecule. Surprisingly, this procedure had no detectable effecton the stability and immunoreactivity of the antibody. There are amaximum number of intrachain disulphide sites which can be exploiteddepending on the antibody subclass. A branched chain hydrazone linkerhas not also been described to exploit this binding site further, bydoubling the number of drug molecules which can be chemically attached(King, et al., (1999) Bioconjug. Chem. 10:279-288).

Antibody fragments have also been used in a number of drug targetingstudies. From a conjugation viewpoint the Fab′ fragment is particularlyconvenient, bearing a single free sulphydryl group which can be readilyused for attachment to drugs or other macromolecules (Hashida, et al.,(1984) J. Appl. Biochem. 6:56-63).

One of the simplest methods of attachment is the use of peptide bondforming reagents such as carbodiimides or active esters which have beenused to attach carboxyl-bearing drug such as methotrexate (MTX). Earlywork with polylysine conjugates, has shown that biodegradableMTX-poly(-_(L)-lysine) shows some cytotoxicity, but thatMTX-poly(-_(D)-Lysine) is non-toxic, suggesting that free drug isreleased through cleavage of the polymer (Ryser, et al., (1980) Cancer45:1207-1211). In that case we would also expect biological moleculessuch as albumin and immunoglobulins to be cleaved to release free drug.Both MTX-immunoglobulin (Kanellos, et al., (1985) J. Natl. Cancer Inst.75:319-332) and MTX-HSA-Immunoglobulin (Garnett, et al., (1983) Int. J.Cancer 31:661-670) conjugates have been reported using this principal.These reactions do not cleanly produce a single product, a mixture oflabile ester and stable amide bonds being formed (Endo, et al., (1988)Cancer Res. 48:3330-3335 and Hudecz, et al., (1992) Biomed. Chromatogr.6:128-132). The ester-linked material can form up to 24% of theconjugated drug, and is gradually released from the conjugate byhydrolysis in storage at 4° C. over 20 days (Hudecz, et al., (1992)Biomed. Chromatogr. 6:128-132). The inhibition of conjugate cytotoxicityby ammonium chloride, and the lysosomal protease inhibitors leupeptinand E64 (Garnett, et al., (1985) Anti-Cancer Drug Design 1:3-12)suggested that the free drug was being released through degradation in alysosomal compartment. However, detailed studies on the release of MTXfrom HSA-MTX conjugates (Fitzpatrick et al., (1995) Anti-Cancer DrugDesign 10:11-24) have shown that the amount of low-molecular weight drugreleased by rat liver tritosomes is very low (5.6% in 55 h). Further,only about 10% of the material released was free MTX, the rest beingamino acids are not readily cleaved from the drug enzymically and aresignificantly less cytotoxic than unmodified methotrexate (Rosowsky, etal., (1984) J. Med. Chem. 27:888-893). Conjugates designed to releasethese amino acid derivatives of MTX were also less cytotoxic thanconjugates releasing free MTX. Release of MTX and MTX derivatives fromantibody-MTX conjugates was much lower, estimated to be <0.05% over thesame period, which was attributed to both the low substitution ratio formethotrexate (Rosowsky, et al., (1984) J. Med. Chem. 27:888-893) and thepoor degradation of the Fab region of antibodies by tritosomes(Schneider, et al., (1981) J. Cell. Biol. 88:380-387). A linker whichspecifically releases free drug from conjugate is therefore a vitalcomponent of targeted drug conjugates. Various types of linkage havebeen reported.

Aldehyde/Schiff base linkages may also be used to link therapeuticagents to antibodies or other localizing agents. Sugar residues withvicinal hydroxyl groups can be converted into aldehyde groups byoxidation with periodate (O'Shanessy, et al., (1984) Immunol. Lett.8:273-277). This enables sugar residues in polysaccharides such asdextran, or sugar moieties in drugs, e.g., nucleoside sugar groups influorouridine, or the amino sugar in daunomycin to have a more usefulaldehyde group inserted. Aldehydes will readily react with hydrazides toform a hydrazone, or with amines to form a Schiff base. The Schiff baseitself is relatively unstable (Cordes, et al., (1963) J. Am. Chem. Soc.85:2843-2848), and can reform its starting materials so is usuallyreduced with a reagent such as sodium borohydride or sodiumcyanoborohydride to stabilize the linkage. Dialdehydes such asglutaraldehyde can also be used to crosslink between drug andmacromolecule, or between macromolecules, but in this case the linkageof the products is usually irreversible (Wong, (1991) CRC Press BocaRaton, Fla. 101-102). The use of glutaraldehyde as a cross-linking agentalso has the disadvantage that it is a homobifunctional reagent, sounwanted cross-links and aggregates can be readily generated.

Sulphydryl linkages may also be used to link therapeutic agents toantibodies or other localizing agents. Disulphide linkages are foundconnecting the chains of plant toxins, and are essential for theiractivity (Masuho, et al., (1982) J. Biochem. 91:1583-1591) so that theenzymic A chain can dissociate from the binding chain and enter thecytoplasm. The necessity of this linkage suggests that this is apossible way of releasing drugs from antibody or polymer molecules inintracellular compartments. Conjugates of methotrexate topoly(_(D)-lysine) through a disulphide linkage were shown to becytotoxic (Shen, et al., (1985) J. Biol. Chem. 260:10905-10908).Cleavage of these conjugates was shown to occur initially at the cellsurface, but did not take place within the endosomal or lysosomalcompartments (Feener, et al., (1990) J. Biol. Chem. 265:18780-18785). Itwas suggested that the Golgi apparatus was the most likely site ofcleavage. Although cleavable intracellularly, this linkage was shown tobe unstable in the circulation, and hindered disulphide bonds havetherefore been developed to reduce this problem (Thorpe, et al., (1987)Cancer Res. 47:5924-5931 and Worrell, et al., (1986) Anti-Cancer DrugDesign 1:179-188). For conjugates where a stable, chemically convenientcoupling via a sulphydryl group is required a thioether bond is morestable and can be produced through the use of maleimide (Hashida, etal., (1984) J. Appl. Biochem. 6:56-63 and Lau, et al., (1995) Bioorg.Med. Chem. 3:1299-1304) or iodacetate (Rector, et al., (1978) J.Immunol. Methods 24:321-336) coupling reagents.

Acid-labile linkages may also be used to link therapeutic agents toantibodies or other localizing agents. Chemically labile linkages couldbe used to release drug in the presence of more acid conditions. Theseconditions can occur either in the tumour environment which is reportedto be 0.5-1 pH unit more acidic than health tissue and blood (Lavie, etal., (1991) Cancer Immunol. Immunther. 33:223-230 and Ashby (1966)Lancet ii:312-315), or during passage through the endosomal/lysosomalcompartment, where pH of 6-6.8 and 4.5-5.5, respectively, can be found.The major drawback to the use of an acid-labile linkage is that this isa rate-dependent phenomenon, where the rate of cleavage is proportionalto pH: a 10-fold difference in rate can be expected for each pH unitdecreased. This means that the hydrolysis will always be a compromisebetween a fast rate at low pH in the intracellular compartment, and aslow rate for serum stability.

The first acid sensitive linker described was the cis-aconityl linkagedescribed by Shen and Ryser (Shen, et al., (1981) Biochem. Biophys. Res.Commun 102:1048-1054. Daunomycin was first reacted with cis-aconiticanhydride, which was then subsequently coupled to poly-lysine using awater-soluble carbodiimide. This linkage was reported to have ahalf-life of less than 3 h at pH 4 and greater then 96 h at pH 6.However, only about 50% of the drug was released from an affi-gelmatrix. This may be due to inappropriate binding of the gel matrix tothe remaining cis-carboxyl group responsible for the acid-sensitiverelease from this linker. The optimum conditions for the use of thisreagent have been described in detail by (Hudecz, et al., (1990)Bioconjug. Chem. 1:197-204). An improved version of this releasemechanism has also been described by (Blattler, et al., (1985)Biochemistry 24:1517-1524), as a heterobifunctional agent for couplingof toxin molecules. In this method, the conjugation through the thirdcarboxyl of the cis-aconitate has been replaced with a specificmaleimido group which will eliminate the possibility of inactivation ofthe acid release properties of the cis-carbonyl group.

A range of acid-sensitive homo- and heterobifunctional agents originallyprepared to give acid-sensitive release for toxin immunoconjugates havebeen described by Srinivasachar, based on ortho esters, acetals andketals (Srinivasachar, et al., (1989) Biochemistry 28:2501-2509). Thesecould also potentially be of use for constructing chemoimmunoconjugates.These reagents vary in their rate of hydrolysis at the pH found inintracellular compartments. Hydrazone linkages may also be used to linktherapeutic agents to antibodies or other localizing agents.

Hydrazide derivatives are also acid labile and have been used to produceboth vindesine and adriamycin conjugates (Laguzza, et al., (1989) J.Med. Chem. 32:548-555 and Greenfield, et al., (1990) Cancer Res.50:6600-6607). In the former, (Laguzza, et al., (1989) J. Med. Chem.32:548-555) vindesine was first reacted with hydrazine, and thehydrazide derivative then reacted with the oxidised sugar residue ofantibody. In the adriamycin conjugate (Greenfield, et al., (1990) CancerRes. 50:6600-6607), an SPDP hydrazine derivative, was prepared which wasreacted with a thiolated antibody. In both of these conjugateslow-molecular weight drug was released under acid conditions. In theformer case, up to about 30% vinca hydrazide was released at pH 5.3 over7 days at 37° C., in the latter case, unmodified adriamycin was releasedrapidly from the conjugate at pH 4.0-5.5. A study of different hydrazonederivatives of adriamycin has been reported (Kaneko, et al., (1991)Bioconjug. Chem. 2:133-141), which show that the acid instability of thevarious linkers is acylhydrazide>semicarbazide>carbonic aciddihydrazide>thiosemicarbazide>hydrazine carboxylate=arylhydrazide, allreleasing adriamycin as the only product. With the exception of thearylhydrazide, all of these compounds were stable at pH 7.4. The acylhydrazine released 85% of the theoretical amount of drug at pH 5.0, 37°C. in 3 h, and when conjugated to an anti-transferrin receptor antibody,was nearly as cytotoxic as free adriamycin. A maleimidocaproylhydrazonederivative has also been synthesized to provide a thioether-linkedconjugate which is more stable in serum, and which can be readilycoupled to reduced intrachain disulphide groups in antibodies(Firestone, et al., (1996) BR96-Dox, J. Control 39:251-259). Furtherlong-chain arylhydrazide linkers for conjugation of anthracyclines havebeen described by (Lau, et al., (1995) Bioorg. Med. Chem. 3:1299-1304).

Enzymically degradable linkers may also be used to link therapeuticagents to antibodies or other localizing agents. The gold standard forattaching and releasing drugs from macromolecules is a linker which isstable in serum but can be cleaved intracellularly by specific enzymes.Linkers of this type have been described containing a variety of aminoacids. Some of these linkers have been used in targeted drug conjugateswith antibodies, but others only in polymer-drug conjugates. Cleavableamino acid pro-drugs of daunomycin (Dau) were first produced by Levinand Sela (Levin, et al., (1979) FEBS Lett. 98:119-122), although thesewere designated as low-molecular weight pro-drugs. The first systematicstudies investigating amino acid sequences and lengths for lysosomaldigestion were reported by (Masquelier et al. (1980) J. Med. Chem.23:1166-1170). These studies identified an Ala-Leu-Dau derivative whichcould be converted back to the free drug by lysosomal hydrolases in 2 h.The activity was ascribed to a lysosomal dipeptidyl aminopeptidase.While these dipeptide derivatives were much less potent than Dau invitro, they showed greater potency in vivo (Baurain, et al., (1980) J.Med. Chem. 23:1171-1174). Further work reported by this group (Trouet,et al., (1982) Proc. Natl. Acad. Sci. USA 79:626-629) resulted inconjugates in which daunorubicin was linked to succinylated serumalbumin by a spacer arm of one to four amino acids. A minimum tri orpeptide spacer was found to be essential for good release of drug. Arelease of 75% of free drug was achieved in 8 h with an albuminconjugate with an Ala-Leu-Ala-Leu-Dau linkage, which was stable in thepresence of serum (only 2.5% drug released in 24 h). No drug wasreleased by lysosomal enzymes from Dau conjugated to succinylated serumalbumin without a peptide spacer.

Another tetrapeptide spacer was derived from a long collaborationbetween Duncan and Kopecek, in which the release of p-nitroaniline as amodel drug from poly[N-(2-hydroxypropyl)methacrylamide] co-polymers wasinvestigated (described in Duncan [(Duncan, (1986) CRC Crit. Rev.Biocompat. 2:127-145)]). These studies resulted in a greaterunderstanding of lysosomal enzyme specificity and the development of aGly-Phe-Leu-Gly-Dau linker which released 80% of bound p-nitroanilineover a 50-h incubation period. Daunomycin was subsequently coupled tothe polymer delivery systems (Duncan, et al., (1987) Br. J. Cancer55:165-174) and as antibody carrier drug conjugates.

A tetrapeptide spacer has been incorporated into monoclonalantibody-methotrexate conjugates by (Umemoto, et al., (1989) Int. J.Cancer 43:677-684). This is a MTX-Leu-Ala-Leu-Ala-hydrazide linker basedon the tetrapeptide described by Trouet. However, in Trouet's study theDau was attached to the C-terminal of the peptide, and in this conjugateMTX was attached to the N terminal of the peptide. In addition there isalso a hydrazide incorporated into the linkage which may give someacid-sensitive release of the drug-linker part of the conjugate. Nostudies were reported on the effect of lysosomal enzymes on this linker,and what products were released, nor the rate of release of products.However, these linkers gave a substantial increase in efficiency of theconjugate compared to directly linked MTX, and release was shown byinhibitors such as leupeptin to be lysosomally mediated.

The development of a further tetrapeptide spacer for an HAS carriermolecule has been described by (Fitzpatrick, et al., (1995) Anti-CancerDrug Design 10:1-9). An appropriate spacer was developed using alysosomal enzyme degradation system, where attachment of the terminalresidue of the peptide chain to an ε-amino lysine residue was used as amodel for conjugation to protein. Using this system it was shown that avariety of amino acids coupled to the carboxyl groups of MTX couldrelease free drug, and the rate of release of free drug was dependent onthe length of the spacer, a tetrapeptide spacer giving about 90% releaseof free drug. Conjugation of the MTX-tetrapeptide to HAS further reducedthe rate of release of free drug to about 30% over 48 h. Theseexperiments show that the tetrapeptide spacer is not just to overcomesteric constraints of a polymer molecules, but also relate to theefficiency of binding of the cleavage site to the enzyme active site.

An efficient and general method for linking anthracyclines to peptidesby an oxime linkage has been described by (Ingallinella, et al., (2001)Bioorg. Med. Chem. Lett. 11:1343-1346; however, no immunoconjugates werereported using this linkage.

Generally the simplest way of producing an immunoconjugate is to couplethe drug directly to the antibody. This may involve a direct linkagebetween the functional group of the drug, and one of the functionalgroups on the antibody, or alternatively may involve the interpositionof a linker or spacer group between these two parts of the conjugate. Alinker group may be used merely to make the chemistry of the couplingpossible, but may have the second function of allowing a specific typeof release of the drug. If the release is mediated by an enzyme, locatedeither intra- or extracellularly, the group may be termed a spacergroup, its purpose being to allow sufficient space, or reduce stericconstraints so that the enzyme can access the relevant bond adequately.

Methotrexate was one of the first cytotoxic drugs to be linked toantibodies. In these early studies using immune sera (Marthé, et al.,(1958) C.R. Acad. Sci. 246:1626-1628 and Burstein, et al., (1977) J.Med. Chem. 20:950-952) with coupling either through diazotization ormixed anhydride procedures. Both of these procedures resulted intherapeutically active conjugates in mouse models.

The conjugates that have been produced have been documented in manyreviews (e.g., Magerstädt (1991) CRC Press Boca Raton, Fla. 77-215;Dubowchik, et al., (1999) Pharmacol. Ther. 83:67-123; and Pietersz, etal., (1994) Adv. Immunol. 56:301-387). Early work withvinblastine-antibody conjugates used a variety of methods forconjugating drug to antibody, with some reports showing increasedcytotoxicity of conjugate compared to free drug (Johnson, et al., (1981)Br. J. Cancer 44:372). Clinical studies on vinblastine conjugates havebeen reported. The first of these studies involved a conjugate with themurine monoclonal antibody KS1/4, using a hemisuccinate derivative ofDAVLB rather then the optimized DAVLBHY (Schneck, et al., (1990) Clin.Pharmacol. Exp. 47:36-41).

The two main anthracyclines used in antibody conjugates are daunomycin(synonymous with daunorubicin) and adriamycin (synonymous withdoxorubicin), differing in only the terminal C14 of the side chain,which is a methyl group in the former and a less hydrophobic methoxygroup in the latter. Daunomycin (Dau) is reported to be more cytotoxicthan doxorubicin (Dox). Idarubicin and epirubicin are slightly morecytotoxic derivatives, with an improved toxicity profile compared to Dauor Dox (Arcamone, (1985) Cain Memorial Aware Lecture, Cancer Res.45:5995-5999). Morpholinodoxorubicin and cyanomorpholino doxorubicinwere reported to be highly cytotoxic derivatives (Newman, et al., (1985)Science 228:1544-1546).

Immunoconjugate preparations of anthracyclines (Dau and Dox) toimmunoglobulin assessed (Hurwitz, et al., (1975) Cancer Res.35:1175-1181): (1) periodate oxidation of the sugar moiety of the drug,conjugation to the lysyl groups of antibody and subsequent reduction ofthe Schiff base reduction, (2) glutaraldehyde coupling between the sugaramino group and lysyl groups of antibody. Drug activity was bestpreserved with glutaraldehyde activity, but both periodate-oxidised andglutaraldehyde-linked conjugates showed good activity against targetcells. The periodate-oxidised conjugates were assessed in more detail(Levy, et al., (1975) Cancer Res. 35:1182-1186) and shown to retainabout 50% of the activity of free drug and have specificity in acytotoxicity assay involving a brief exposure to immunoconjugate. Theantitumour effects of the conjugate on PC5 B-cell leukemia were betterthan the free drug.

Idarubicin (Ida) immunoconjugates were prepared from 14-bromo-idarubicinwith anti-ly2.1 antibodies with an MR of 1-5 (Pietersz, et al., (1988)Cancer Res. 48:926-931).

Conjugates showed selective cytotoxicity that was less active than freedrug on E3 target cells (IC₅₀=430 and 120 nM, respectively). Anti-tumouractivity was shown on E3 thymoma xenografts by reduction of tumourgrowth rate which was greater than that produced by Ida alone. Furtherstudies using IDA immunoconjugates prepared from anti-CD19 antibody gaveactivities of 240 nM for immunoconjugate compared to 12 nM for free Ida(Rowland, et al., (1993) Cancer Immunol. Immunother. 37:195-202).

The anthracyclines have been a popular choice of drug for targeteddelivery. The morpholino group makes the sugar amino group commonly usedfor conjugation unavailable so linkers via the C13 on the side chainwere used.

Immunoconjugates with 5-fluoro-2′-deoxyuridine (FUDR) were constructedby reacting an active ester derivative of succinylated FUDR withanti-ly1.2 monoclonal antibody giving a conjugate with drug to antibodyMR of 7-9 (Krauer, et al., (1992) Cancer Res. 52:132-137). On theantigen-positive E3 cell line succinylated FUDR, and immunoconjugateboth gave similar cytotoxicities (IC₅₀=5 and 3 nM, respectively) whichwere about 10-fold lower than free drug (0.4 nM). In vivo a greaterinhibition of E3 thymoma tumour growth was seen with the immunoconjugatethan with an equivalent amount of free drug.

A taxol immunoconjugate has been reported by Guillemard and Saragovi(Guillemard, et al., (2001) Cancer Res. 61:694-699). Taxol was firstmodified with glutaric anhydride, to give a cleavable ester linkage tothe drug, and then conjugated directly to antibody using carbodiimide.Immunoconjugate to anti-mouse and anti-rat IgG were made with an MR ofdrug to antibody of 1. Cytotoxicity tests appeared to show thatconjugates were more cytotoxic than free drug. In vivo, immunoconjugateshowed a small but significant reduction of tumour growth onneuroblastoma xenografts.

Antibody concentration is an important determinant of the rate of druguptake; therefore, if more drug molecules can be conjugated per antibodymolecule, cytotoxicity should increase. However, as the loss of antibodybinding activity is the rate-limiting factor in the number of drugmolecule, the use of carrier molecules for targeted drug conjugatesoffers a solution to this problem. A number of types of conjugate havebeen explored, mostly using dextran, human serum albumin orhydroxypropylmethacrylamide (HPMA) as the carrier molecules, and usingthe drugs doxorubicin or methotrexate. The earliest carrier conjugatewas of phenylene diamine mustard to antibody via a polyglutamic acidcarrier, showing a 45:1 molar substitution ratio (Rowland, et al.,(1975) Nature 255:487-488).

Another solution to the difficulties of delivering sufficient drugmolecules to kill cancer cells is to use more potent drugs, whichrequire fewer molecules of drug to kill a cell. A number of thesemolecules have been discovered and investigated as potential anti-tumouragents. These include: CC-1065-like alkylating agents such asDuocarmycin; Enediynes, including the dynemicins, thecalicheamicins/esperamicins, and the chromoproteins (Borders, et al.,(1994) Marcel Dekker New York) (e.g., Neocarzinostatin, andCalicheamicin), and Macrolide antibiotics such as Geldanamycin andmaytansine.

Application of tumor-targeted cytotoxic therapeutics has been usedsuccessfully with many tumor antigens. For example, Gemtuzumabozogamicin, used to treat acute myelogenous leukemias (AMLs), iscomposed of an anti-CD33 antibody attached to calicheamicin, anantitumor chemotherapeutic agent. The CD33 antigen is present on myeloidprecursors, but not on hematopoietic stem cells, so the CD33-targetedtherapeutic is selective for AML cells while sparing critical normalcell types. Binding of this agent to the CD33 antigen present of AMLcells results in the formation of a complex that is internalized intothe cell. After internalization, the antitumor moiety of this agent isreleased into the myeloid cells and causes cell death (Naito et al.(2000) Leukemia 14: 14636-43). This drug has been approved by the FDAfor treatment of relapsed or refractory AML in patients over 60 yearsold (Abou-Jawde et al. (2003) Clin. Therap. 25: 2121-37). Furthermore,gemtuzumab ozogamicin has shown benefit in treating recurrent AMS and,as such, may help many patients.

Immuntoxins

The therapeutic agent or drug moiety is not to be construed as limitedto classical chemical or radiological therapeutic agents. For example,the drug moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, in additionto toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin, other proteins with biological activity such as tumor necrosisfactor, alpha-interferon, beta-interferon, nerve growth factor, plateletderived growth factor, tissue plasminogen activator, a thrombotic agentor an anti-angiogenic agent, e.g., angiostatin or endostatin; or,biological response modifiers such as, for example, lymphokines, IL-1,IL-2, IL-4, IL-5, IL-6, IL-7, 11-8, 11-9, 11-10, IL-12, IL-15,granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Immunotoxins contain a ligand such as a growth factor, monoclonalantibody, or fragment of an antibody which is connected to a proteintoxin. After the ligand subunit binds to the surface of the target cell,the molecule internalizes and the toxin kills the cell. Bacterial toxinswhich have been targeted to cancer cells include Pseudomonas exotoxinand diphtheria toxin, which are well suited to forming recombinantsingle-chain or double-chain fusion toxins. Plant toxins include ricin,abrin, pokeweed antiviral protein, saporin and gelonin, and havegenerally been connected to ligands by disulfide-bond chemistry.Immunotoxins have been produced to target hematologic malignancies andsolid tumors via wide variety of growth factor receptors and antigens.

The goal of immunotoxin therapy is to target a cytotoxic agent to cellsurface molecules which will internalize the cytotoxic agent and resultin cell death. Since immunotoxins differ greatly from chemotherapy intheir mode of action and toxicity profile, immunotoxins provide improvedsystemic treatment of tumors.

Immunotoxins can be simply defined as proteins containing a toxin and anantibody. Toxins reviewed here include catalytic proteins produced byplants or bacteria which kill target cells. While the term ‘immunotoxin’generally refers to a toxin targeted by either an intact IgG, an Fabfragment or an Fv fragment, toxins targeted by growth factors or otherligands are also referred to as ‘chimeric toxins’. In some immunotoxinsor chimeric toxins, the linkage between the ligand and the toxin is madechemically, and the proteins may be referred to as ‘chemicalconjugates’. Otherwise, when the linkage is a peptide bond produced bygenetic engineering, the proteins are referred to as ‘recombinanttoxins’ or ‘fusion toxins’. Finally, a select group of immunotoxinscontain an Fv sequence fused to the toxin, and these proteins, beingboth immunotoxins and recombinant toxins, are often referred to as‘recombinant immunotoxins’.

Protein toxins are well suited because of their extreme potency. It hasbeen shown that one or a few molecules of protein toxins can kill a cellwhen injected into the cytoplasm (see Yamaizumi, et al., (1978) Cell15:245-250 and Eiklid, et al., (1980) Exp. Cell Res. 126:321-326).

Plant toxins exist in nature as holotoxins and hemitoxins. Holotoxins(also referred to as class II ribosome in activating proteins) includericin, abrin, misdetoe lectin and modeccin, which contain a bindingdomain disulfide-bonded to an enzymatic domain. Hemitoxins, such aspokeweed antiviral protein (PAP), saporin and gelonin contain anenzymatic but no binding domain.

To make immunotoxins, plant toxins are generally conjugated chemicallyto ligands (see e.g., Kreitman, et al., (1998) Adv. Drug Del. Rev.,31:53-88).

Two bacterial toxins generally used to make immunotoxins includePseudomonas exotoxin (PE), made by Pseudomonas aeruginosa, anddiphtheria toxin (DT), made by Corynebacterium diphtherae. Both PE andDT catalytically ADP ribosylate EF-2 in the cytosol (see Carroll et al.,(1987) J. Biol. Chem. 262:8707-8711; Uchida et al., (1972) Science175:901-903; and Uchida et al., (1973) J. Biol. Chem. 248:3838-3844).

Mutated and truncated forms of DT and PE may also be used (see Kreitman,et al., (1998) Adv. Drug Del. Rev., 31:53-88). For example, mutanttoxins will be designed in which the binding domain of the toxin wasdeleted or made non-functional by mutation. In the case of DT this couldbe done chemically by treating the toxin with trypsin and purifying theA-chain (see Masuho, et al., (1979) Biochem. Biophys. Res. Commun.90:320-326). Recombinant toxins which are full-length and containmutated binding domains include PE^(4E), containing glutamate replacingbasic residues at positions 57, 246, 247 and 249 of PE, and CRM107,containing phenylalanines replacing a leucine at position 390 and serineat position 525 of DT (see Greenfield, et al., (1987) Science238:536-539 and Chaudhary, et al., (1990) J. Biol. Chem.265:16306-16310).

A wide variety of trace immunotoxins and recombinant toxins have beenmade and tested against malignant target cells (see Kreitman, et al.,(1998) Adv. Drug Del. Rev., 31:53-88).

The next generation of immunotoxin include recombinant toxins,immunotoxins containing an antibody or antibody fragment like an Fab′chemically conjugated to a toxin have several disadvantages. Firstly,their large size (100-200 kDa), often results in reduces tumorpenetration. Secondly, for conjugation to antibodies, toxins such asPE40 and PE38 must be derivatized with reagents which modify the lysineresidues, many of which are near the carboxyl terminus. Similarly, theantibody may require derivatization of lysine residues within theantigen binding domains. The resulting immunotoxins are therefore aheterogeneous mixture with respect to sites of attachment of theantibody and toxin, as well as the number of toxin and antibodycomponents per immunotoxin molecule. Finally, chemical conjugates aredifficult to produce, because the toxin and antibody must be purifiedseparately, conjugated, and then the product repurified.

Toxins can be targeted to cells without chemically conjugating theligand and the toxin if both are connected as one polypeptide unit.

The bacterial toxins PE and DT are optimal for making these fusiontoxins because each toxin contains a proteolytic processing site withina disulfide loop which allows the catalytic domain to separate from therest of the toxin after internalization and translocate efficiently tothe cytosol (see Chiron, et al., 1994) J. Biol. Chem. 269:18167-18176);Fryling, et al., (1992) Infect. Immun. 60:497-502; Ogata, et al., (1992)J. Biol. Chem. 267:25396-25401; and Williams, et al., (1990) J. Biol.Chem. 265:20673-20677).

In 1981 it is reported that the Mab B3/25 was conjugated to truncated DTor RTA and used to inhibit the growth of human melanoma in nude mice(see Trowbridge, et al., (1981) Nature 294:171-173). These and similarimmunotoxins have displayed antitumor activity against a variety ofsolid tumors, including gastrointestinal adenocarcinomas, mesothelioma,cervical cancer and glioblastoma (see Griffin, et al., (1998) J. Biol.Response Mod. 7:559-567; Griffin, et al., (1987) Cancer Res.47:4266-4270; and Martell, et al., (1993) Cancer Res. 53:1348-1353). TheMab HB21 was conjugated to full-length PE and deliveredintraperitoneally to increase the survival of mice harboring humanovarian carcinoma (see FitzGerald, et al., (1986) Proc. Natl. Acad. Sci.USA 83:6627-6630). HB21 as well as its Fab′, (Fab′)₂ and Fv fragmentshave also been conjugated or fused to truncated PE or DT and shown tocause antitumor activity in a variety of models (see Batra, et al.,(1989) Proc. Natl. Acad. Sci. USA 86:8545-8549; Debinski, et al., (1991)Cancer Res. 52:5379-5385; and Batra, et al., (1991) Mol. Cell. Biol.11:2200-2205).

Many of the cell lines that are targets for immunotoxins targeting avariety of antigens are relatively resistant to chemotherapyImmunotoxins have also been made to specifically target cells resistantto multiple chemotherapeutic agents by targeting the p-glycoproteinmolecule which is responsible for increased export of chemotherapeuticagents from cells. The Mab MRK16 conjugated to PE was very cytotoxictoward those cell lines that were most resistant to chemotherapy due toexpression of p-glycoprotein (see FitzGerald, et al., (1987) Proc. Natl.Acad. Sci. USA 84:4288-4292). This immunotoxin also killedmultidrug-resistant carcinoma cells in MDR-transgenic mice (seeMickisch, et al., (1993) J. Urol. 149:174-178). This antibody has alsobeen conjugated to saporin to form an immunotoxin able to purgemultidrug resistant cells from bone marrow (see Dinota, et al., (1990)Cancer Res. 50:291-4294.

Targeted Radiotherapy

Radioisotopes may also be used as cytotoxic agents for vimentin-targetedtherapeutics. Anti-vimentin antibodies of the present invention may becoupled to one or more therapeutic agents. Suitable agents in thisregard include radionuclides. Suitable radionuclides include ⁹⁰Y, ¹²³I,¹²⁵I, ¹³¹I, ¹⁸⁶Re, ²¹¹At or ²¹²Bi. Carriers specific for radionuclideagents, to facilitate attachment to the vimentin targeting agent,include radiohalogenated small molecules and chelating compounds. Forexample, U.S. Pat. No. 4,735,792 discloses representativeradiohalogenated small molecules and their synthesis. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide. For example, U.S. Pat. No.4,673,562, to Davison et al. discloses representative chelatingcompounds and their synthesis.

An ideal radioligand therapy agent would accumulate selectively intarget cells. The effectiveness of radiotherapy is due to thedestruction of dividing cells resulting from radiation-induced damage tocellular DNA (see, e.g., W. D. Bloomer et al., (1977) “TherapeuticApplication of Iodine-125 Labeled Iododeoxyuridine in an Early AscitesTumour Model,” Current Topics in Radiation Research Quarterly12:513-25). In both therapeutic and imaging applications, any unbound,circulating radioligand is rapidly cleared by excretory systems, whichhelps protect normal organs and tissues. The radioligand may also bedegraded by body processes which will increase the clearance of the freeradioisotope (see G. A. Wiseman et al. (1995) “Therapy of NeuroendocrineTumors with Radiolabelled MIBG and Somatostatin Analogues,” Seminars inNuclear Medicine, vol. XXV, No. 3, pp. 272-278).

Radioisotopes most suitable for therapeutic treatment includeAuger-electron-emitting radioisotopes, e.g. ¹²⁵I, ¹²³I, ¹²⁴I, ¹²⁹I,¹³¹I, ¹¹¹In, ⁷⁷Br, and other radiolabeled halogens. The choice of asuitable radioisotope can be optimized based on a variety of factorsincluding the type of radiation emitted, the emission energies, thedistance over which energy is deposited, and the physical half-life ofthe radioisotope. In certain instances, the radioisotopes used are thosehaving a radioactive half-life corresponding to, or longer than, thebiological half-life of the vimentin-targeted therapeutic. For example,in certain examples the radioisotope has a half-life between about 1hour and 60 days, preferably between 5 hours and 60 days, morepreferably between 12 hours and 60 days. ¹²⁵I has an advantage overother emitters that produce high-energy gamma rays (i.e., ¹¹¹In and¹³¹I) which require inpatient hospitalization and isolation ¹²⁵I willallow the development of outpatient-based treatments due to the limitedamounts of radiation that escapes the body.

Radiolabeled therapeutics have typically been administered byintravenous, bolus injection (see, e.g., H. P. Kalofonos et al.,(1989)“Antibody Guided Diagnosis and Therapy of Brain Gliomas usingRadiolabeled Monoclonal Antibodies Against Epidermal Growth FactorReceptor and Placental Alkaline Phosphatase” The Journal of NuclearMedicine vol. 30, pp. 163-645; I. Virgolini et al., (1994) “VasoactiveIntestinal Peptide-Receptor Imaging for the Localization of IntestinalAdenocarcinomas and Endocrine Tumors” The New England Journal ofMedicine, vol. 331, pp., 1116-21; G. A. Wiseman et al., (1995) “Therapyof Neuroendocrine Tumors with Radiolabelled MIBG and SomatostatinAnalogues” Seminars in Nuclear Medicine, vol. XXV, no. 3, pp. 272-78; S.W. J. Lamberts et al., (1990) “Somatostatin-Receptor Imaging in theLocalization of Endocrine Tumors” The New England Journal of Medicinevol. 323, pp. 126-49; E. P. Krenning et al. (1992) “SomatostatinReceptor Scintigraphy with Indium-111-DTPA-D-Phe-1-Octreotide in Man:Metabolism, Dosimetry and Comparison with Iodine-123-Tyr-3-Octreotide”The Journal of Nuclear Medicine vol. 33, pp. 652-58; E. P. Krenning etal. (1989) “Localisation of Endocrine-Related Tumours withRadioiodinated Analogue of Somatostatin,” The Lancet vol. 1989, no. 1,pp. 242-244.

Targeted Gene Therapy

Gene vectors may also be used as cytotoxic agents for vimentin-targetedtherapeutics. For example, a gene vector encoding an antibody gene (orfragment thereof) inside the tumor cell. The transgene expressionproduct binds intracellular proteins, e.g., those derived fromoncogenes, and thereby down-regulates oncogenic protein expression.Targeted gene therapy may be facilitated by the use of bifunctionalcrosslinkers to target adenoviral and retroviral vectors, by insertingshort targeting peptides and larger polypeptide-binding domains into thecoat protein of a number of different viral vectors, and by the use ofreplication-competent vectors (see Wand and Liu (2003) Acta Biochimicaet Biophysica Sinica 35(4): 311-6). Other non-viral therapeutic agents,including DNA complexes and bacterial vehicles, have also beendeveloped. Gene therapy methods for vimentin-targeted compositions andmethods of the invention may be adapted from gene therapy methods knownin the art or adapted from U.S. Pat. Nos. 5,871,726, 5,885,806,5,888,767, 5,981,274, 6,207,426, 6,210,708, 6,232,120, 6,498,033,6,537,805, 6,555,107, and 6,569,426.

In one approach, targeted replicative or non-replicative viral vectorsmay be used to deliver the gene therapeutic. For example, andoviral genetherapy vectors have been adapted for the targeting of neoplastic cells(see Rots, et al. (2003) Journal of Controlled Release 87: 159-165).Selective targeting of adenovirus vectors limits the inflammatory andimmune response against the viral vector and decreases the toxicity ofthe treatment because lower doses of virus can be used. Adenoviralinfection is normally initiated by the binding of target cells by theC-terminal part of the adenovirus fiber protein, termed know, and theprimary cellular receptor, coxsackie B virus and adenovirus receptor(CAR). After this step, entry of the virus into the cell occurs viainteraction of the RGD (arg-gly-asp) sequence of viral penton baseprotein and cellular integrins. Selective targeting of adenovirusvectors can be achieved. Linking (e.g., conjugation) of avimentin-specific antibody to the adenoviral vector will target theresulting construct to vimentin-expressing neoplastic, MDR neoplasticand damaged (e.g. pathogen infected) cells. For example, this strategyhas been successfully adapted to target adenovirus to the EGP-2 antigenpresent on tumor cells (Heiderman et al. (2001) Cancer Gene Ther. 8:342-51) by conjugating a neutralizing anti-fiber protein antibody to anantibody against the Epithelial Cell Adhesion Molecule (EGP-2). Theresulting EGP-2 adenovirus was targeted to cancer cells expressingEGP-2, and infection was shown to be independent of CAR. Anotherstrategy is to use bispecific antibodies to bridge cell surface vimentinto the therapeutic gene delivery vector (e.g., adenoviral vector) (see,e.g., Haisma et al. (2000) Cancer Gene 7: 901-4; Grill et al. (2001)Clin. Cancer Res. 7: 641-50; Krasnykh et al. (1998) J. Virol. 72:1844-52; and van Beusechem et al. (2000) Gene Ther. 7: 1940-46).

In general, the terms “viral vectors” and “viruses” are usedinterchangeably herein to refer to any of the obligate intracellularparasites having no protein-synthesizing or energy-generating mechanism.The viral genome may be RNA or DNA contained with a coated structure ofprotein of a lipid membrane. The terms virus(es) and viral vector(s) areused interchangeably herein. The viruses useful in the practice of thepresent invention include recombinantly modified enveloped ornon-enveloped DNA and RNA viruses, preferably selected frombaculoviridiae, parvoviridiae, picornoviridiae, herpesviridiae,poxyiridae, or adenoviridiae. The viruses may be naturally occurringviruses or their viral genomes may be modified by recombinant DNAtechniques to include expression of exogenous transgenes and may beengineered to be replication deficient, conditionally replicating orreplication competent. Chimeric viral vectors which exploit advantageouselements of each of the parent vector properties (See e.g., Feng, etal., (1997) Nature Biotechnology 15:866-870) may also be useful in thepractice of the present invention. Minimal vector systems in which theviral backbone contains only the sequences need for packaging of theviral vector and may optionally include a transgene expression cassettemay also be produced according to the practice of the present invention.Although it is generally favored to employ a virus from the species tobe treated, in some instances it may be advantageous to use vectorsderived from different species that possess favorable pathogenicfeatures. For example, equine herpes virus vectors for human genetherapy are described in WO98/27216 published Aug. 5, 1998. The vectorsare described as useful for the treatment of humans as the equine virusis not pathogenic to humans. Similarly, ovine adenoviral vectors may beused in human gene therapy as they are claimed to avoid the antibodiesagainst the human adenoviral vectors. Such vectors are described in WO97/06826 published Apr. 10, 1997.

The term “replication deficient” refers to vectors which are incapableof replication in a wild type mammalian cell. In order to produce suchvectors in quantity, the producer cell line must be cotransfected with ahelper virus or modified to complement the missing functions. Forexample, 293 cells have been engineered to complement adenoviral E1deletions allowing propagation of the E1 deleted replication deficientadenoviral vectors in 293 cells. The term “replication competent viralvectors” refers to a viral vector which is capable of infection, DNAreplication, packaging and lysis of an infected cell. The term“conditionally replicating viral vectors” is used herein to refer toreplication competent vectors which are designed to achieve selectiveexpression in particular cell types while avoiding untoward broadspectrum infection. Such conditional replication may be achieved byoperably linking tissue specific, tumor specific or cell type specificor other selectively induced regulatory control sequences to early genes(e.g. the E1 gene of adenoviral vectors).

In addition to targeting, cell type specificity with viral vectors maybe improved through the use of a pathway responsive promoters driving arepressor of viral replication. The term “pathway-responsive promoter”refers to DNA sequences that bind a certain protein and cause nearbygenes to respond transcriptionally to the binding of the protein innormal cells. Such promoters may be generated by incorporating responseelements which are sequences to which transcription factors bind. Suchresponses are generally inductive, though there are several cases whereincreasing protein levels decrease transcription. Pathway-responsivepromoters may be naturally occurring or synthetic. Pathway-responsivepromoters are typically constructed in reference to the pathway or afunctional protein that is targeted. For example, a naturally occurringp53 pathway-responsive promoter would include transcriptional controlelements activated by the presence of functional p53 such as the p21 orbax promoter. Alternatively, synthetic promoters containing p53 bindingsites upstream of a minimal promoter (e.g. the SV40 TATA box region) maybe employed to create a synthetic pathway-responsive promoter. Syntheticpathway-responsive promoters are generally constructed from one or morecopies of a sequence that matches a consensus binding motif. Suchconsensus DNA binding motifs can readily be determined. Such consensussequences are generally arranged as a direct or head-to-tail repeatseparated by a few base pairs.

Examples of pathway-responsive promoters useful in the practice of thepresent invention include synthetic insulin pathway-responsive promoterscontaining the consensus insulin binding sequence (Jacob, et al. (1995)J. Biol. Chem. 270:27773-27779), the cytokine pathway-responsivepromoter, the glucocorticoid pathway-responsive promoter (Lange, et al.(1992) J. Biol. Chem. 267:15673-80), IL1 and IL6 pathway-responsivepromoters (Won K.-A and Baumann H. (1990) Mol. Cell. Biol. 10:3965-3978), T3 pathway-responsive promoters, thyroid hormonepathway-responsive promoters containing the consensus motif, the TPApathway-responsive promoters (TREs), TGF-beta pathway-responsivepromoters (as described in Grotendorst, et al. (1996) Cell Growth andDifferentiation 7: 469-480). Additionally, natural or synthetic E2Fpathway responsive promoters may be used. An example of an E2F pathwayresponsive promoter is described in Parr, et al. (1997) Nature Medicine3:1145-1149) which describes an E2F-1 promoter containing 4 E2F bindingsites and is reportedly active in tumor cells with rapid cycling.Examples of other pathway-responsive promoters are well known in the artand can be identified in the Database of Transcription RegulatoryRegions on Eukaryotic Genomes accessible through the Internet athttp://www.eimb.rssi.ru/TRRD.

In the certain applications of the invention, the viral vector is anadenovirus. The term “adenovirus” is synonomous with the term“adenoviral vector” and refers to viruses of the genus adenoviridiae.The term adenoviridiae refers collectively to animal adenoviruses of thegenus mastadenovirus including but no limited to human, bovine, ovine,equine, canine, porcine, murine and simian adenovirus subgenera. Inparticular, human adenoviruses includes the A-F sugenera as well as theindividual serotypes thereof the individual serotypes and A-F subgeneraincluding but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5,6, 7, 8, 9, 10, 11 (Ad11A and Ad 11P), 12, 13, 14, 15, 16, 17, 18, 19,19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a,35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Theterm bovine adenoviruses includes but is not limited to bovineadenovirus types 1, 2, 3, 4, 7, and 10. The term canine adenovirusesincludes but is not limited to canine types 1 (strains CLL, Glaxo,RI261, Utrect, Toronto 26-61) and 2. The term equine adenovirusesincludes but is not limited to equine types 1 and 2. The term porcineadenoviruses includes but is not limited to porcine types 3 and 4. Theterm viral vector includes replication deficient, replication competentand conditionally replicating viral vectors.

Particularly useful are vectors derived from human adenovirus types 2and 5. These vectors may incorporate particular modifications to enhancetheir therapeutic potential. For example they may include deletions ofE1a and E1b genes. Certain other regions may be enhanced or deleted toprovide specific features. For example upregulation of the E3 region isdescribed to reduce the immunogenicity associated with human adenoviralvectors administered to human subjects. The E4 region has beenimplicated as important to expression of transgenes from the CMVpromoter, however the E4orf 6 protein has been described as leading tothe degradation of p53 in target cells in the presence of E1b largeprotein (Steegenga, et al. (1998) Oncogene 16:345-347).

The therapeutic gene to be delivered is generally a cytotoxic gene, atumor suppressor gene, a toxin gene, a pro-apoptotic gene, a pro-drugactivating gene, or a cytokine gene. The term “cytotoxic transgene”refers to a nucleotide sequence the expression of which in the targetcell induces lysis or apoptosis of the cell. The term cytotoxictransgene includes but is not limited to tumor suppressor genes, toxingenes, cytostatic genes, pro-drug activating genes, or apoptotic genes.The vectors of the present invention may be used to produce one or moretherapeutic transgenes, either in tandem through the use of IRESelements or through independently regulated promoters.

The term “tumor suppressor gene” refers to a nucleotide sequence, theexpression of which in the target cell is capable of supressing theneoplastic phenotype and/or inducing apoptosis. Examples of tumorsuppressor genes useful in the practice of the present invention includethe p53 gene, the APC gene, the DPC-4 gene, the BRCA-1 gene, the BRCA-2gene, the WT-1 gene, the retinoblastoma gene (Lee, et al. (1987) Nature329:642), the MMAC-1 gene, the adenomatous polyposis coli protein (U.S.Pat. No. 5,783,666), the deleted in colon carcinoma (DCC) gene, theMMSC-2 gene, the NF-1 gene, nasopharyngeal carcinoma tumor suppressorgene that maps at chromosome 3p21.3. (Cheng, et al. (1998) Proc. Nat.Acad. Sci. 95:3042-3047), the MTS1 gene, the CDK4 gene, the NF-1 gene,the NF2 gene, and the VHL gene.

The term “toxin gene” refers to nucleotide sequence, the expression ofwhich in a cell produces a toxic effect. Examples of such toxin genesinclude nucleotide sequences encoding pseudomonas exotoxin, ricin toxin,diptheria toxin, and the like.

The term “pro-apoptotic gene” refers to a nucleotide sequence, theexpression thereof results in the programmed cell death of the cell.Examples of pro-apoptotic genes include p53, adenovirus E3-11.6K, theadenovirus E4orf4 gene, p53 pathway genes, and genes encoding thecaspases.

The term “pro-drug activating genes” refers to nucleotide sequences, theexpression of which, results in the production of protein capable ofconverting a non-therapeutic compound into a therapeutic compound, whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell. An example of a prodrug activating gene isthe cytosine deaminase gene. Cytosine deaminase converts5-fluorocytosine to 5-fluorouracil, a potent antitumor agent). The lysisof the tumor cell provides a localized burst of cytosine deaminasecapable of converting 5FC to 5FU at the localized point of the tumorresulting in the killing of many surrounding tumor cells. This resultsin the killing of a large number of tumor cells without the necessity ofinfecting these cells with an adenovirus (the so-called bystandereffect”). Additionally, the thymidine kinase (TK) gene (see U.S. Pat.No. 5,631,236 and U.S. Pat. No. 5,601,818) in which the cells expressingthe TK gene product are susceptible to selective killing by theadministration of gancyclovir may be employed. The term “cytokine gene”refers to a nucleotide sequence, the expression of which in a cellproduces a cytokine. Examples of such cytokines include GM-CSF, theinterleukins, especially IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20,interferons of the alpha, beta and gamma subtypes especially interferonalpha-2b and fusions such as interferon alpha-2-alpha-1.

Modifications and/or deletions to the above referenced genes so as toencode functional subfragments of the wild type protein may be readilyadapted for use in the practice of the present invention. For example,the reference to the p53 gene includes not only the wild type proteinbut also modified p53 proteins. Examples of such modified p53 proteinsinclude modifications to p53 to increase nuclear retention, deletionssuch as the delta13-19 amino acids to eliminate the calpain consensuscleavage site, modifications to the oligomerization domains (asdescribed in Bracco, et al. PCT published application WO97/0492 or U.S.Pat. No. 5,573,925).

The invention further includes use of gene-targeted non-viral vectors.“Non-viral vector” for use in this aspect of the invention includeautonomously replicating, extrachromosomal circular DNA molecules,distinct from the normal genome and nonessential for cell survival undernon-selective conditions capable of effecting the expression of a DNAsequence in the target cell. Plasmids autonomously replicate in bacteriato facilitate bacterial production. Additional genes, such as thoseencoding drug resistance, can be included to allow selection orscreening for the presence of the recombinant vector. Such additionalgenes can include, for example, genes encoding neomycin resistance,multi-drug resistance, thymidine kinase, beta-galactosidase,dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase.

In order to target the therapeutic gene to neoplastic, MDR neoplasticand damaged (e.g., pathogen-infected) cells, it is advantageous, incertain instances, to incorporate additional elements into non-viralgene delivery systems which facilitate cellular targeting. For example,a lipid encapsulated expression plasmid may incorporate vimentinantibodies or ligands to facilitate targeting. Although a simpleliposome formulation may be administered, the liposomes either filled ordecorated with a desired composition of the invention of the inventioncan delivered systemically, or can be directed to a tissue of interest,where the liposomes then deliver the selected therapeutic/immunogenicpeptide compositions. Vimentin antibodies and ligand for use in thisapplication include antibodies, monoclonal antibodies, humanizedantibodies, single chain antibodies, chimeric antibodies or functionalfragments (Fv, Fab, Fab′) thereof. Alternatively, non-viral vectors canbe linked through a polylysine moiety to a targeting moiety as describedin Wu, et al. U.S. Pat. No. 5,166,320 and U.S. Pat. No. 5,635,383.

Liposomal Formulations

Another strategy that may be employed for vimentin-targeted delivery oftherapeutic agents is the use of immunoliposomes. Immunoliposomesincorporate antibodies against tumor-associated antigens into liposomes,which carry the therapeutic agent or an enzyme that activates anotherwise inactive prodrug (see, e.g., Lasic et al. (1995) Science 267:1275-76). A number of pre-clinical reports have reported successfultargeting and enhanced anti-cancer efficacy with immunoliposomal drugs(Maruyama et al. (1990) J. Pharm. Sci. 74: 978-84); Maruyama et al.(1995) Biochim. Biophys. Acta 1234: 74-80; Otsubo et al. (1998)Antimicrob. Agents Chemother. 42: 40-44; Lopes de Menezes et al. (1998)Cancer Res. 58: 3320-30).

Alternatively, non-antibody vimentin binding agents such as modified LDLmay be used as tumor-specific ligands in targeting liposoomalformulations of therapeutics. For example, folate-coupled liposomes canbe used to target therapeutics to tumors which overexpress the folatereceptor. Folate-coupled liposomes have been successfully delivered tofolate receptor-overexpressing cancer cells in vitro as well as in vivo(Lee and Low (1994) J. Biol. Chem. 269: 3198-204; Lee and Low (1995)Biochim. Biophys. Acta 1233: 134-44; Rui et al. (1998) J. Am. Chem. Soc.120: 11213-18; and Gabizon et al. (1999) Bioconj. Chem. 10: 289-98).Indeed, several pre-clinical reports have described the successfultargeting of liposomal drugs coupled to such ligands (Ichinose et al.(1998) Anticancer Res. 18: 401-4; Yamamoto et al. (2000) Oncol. Rep. 7:107-11; Rui et al. (1998) J. Am. Chem. Soc. 120: 11213-18; and Gabizonet al. (1999) Bioconj. Chem. 10: 289-98). Transferrin has been employedas a targeting ligand to direct liposomal drugs to various types ofcancer cell in vivo (Ishida and Maruyama (1998) Nippon Rinsho 56:657-62; Kirpotin et al. (1997) Biochem. 36: 66-75). PEG-immunoliposomeswith anti-transferrin antibodies coupled to the distal ends of the PEGpreferentially associate with C6 glima cells in vitro and significantlyincreased gliomal doxorubicin uptake after treatment with thetumor-specific long-circulating liposomes containing doxorubicin(Eavarone et al. (2000) J. Biomed. Mater. Res. 51: 10-14).

Methods of forming liposomal micelle/drug formulations are known in theart. For example, therapeutic drug micelles can be formed by combining atherapeutic drug and a phosphatidyl glycerol lipid derivative (PGLderivative). Briefly, the therapeutic drug and PGL derivative are mixedin a range of 1:1 to 1:2.1 to form a therapeutic drug mixture.Alternatively, the range of therapeutic drug to PGL derivative is in theranges 1:1.2; or 1:1.4; or 1:1.5; or 1:1.6; or 1:1.8 or 1:1.9 or 1:2.0or 1:2.1. The mixture is then combined with an effective amount of atleast a 20% organic solvent such as an ethanol solution to form micellescontaining the therapeutic drug. Methods for inclusion of an antibody ortumor targeting ligand into the micelle formulation to produceimmunoliposomes are known in the art and described further below. Forexample, methods for preparation and use of immunoliposomes aredescribed in U.S. Pat. Nos. 4,957,735, 5,248,590, 5,464,630, 5,527,528,5,620,689, 5,618,916, 5,977,861, 6,004,534, 6,027,726, 6,056,973,6,060,082, 6,316,024, 6,379,699, 6,387,397, 6,511,676 and 6,593,308.

As used herein, the term “phosphatidyl glycerol lipid derivative (PGLderivative)” is any lipid derivative having the ability to form micellesand have a net negatively charged head group. This includes but is notlimited to dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoylphosphatidyl glycerol, and dicapryl phosphatidyl glycerol. In oneaspect, phosphatidyl derivatives with a carbon chain of 10 to 28 carbonsand having unsaturated side aliphatic side chain are within the scope ofthis invention. The complexing of a therapeutic drug withnegatively-charged phosphatidyl glycerol lipids having variations in themolar ratio giving the particles a net positive (1:1) neutral (1:2) orslightly negative (1:2.1) charge will allow targeting of differenttissues in the body after administration. However, complexing of atherapeutic drug with negatively charged PGL has been shown to enhancethe solubility of the therapeutic drug in many instances, thus reducingthe volume of the drug required for effective antineoplastic therapy. Inaddition, the complexing of a therapeutic drug and negatively chargedPGL proceeds to very high encapsulation efficiency, thereby minimizingdrug loss during the manufacturing process. These complexes are stable,do not form precipitates and retain therapeutic efficacy after storageat 4° C. for at least 4 months. In order to achieve maximum therapeuticefficacy by avoiding rapid clearance from the blood circulation by thereticuloendothelial system (RES), immunoliposomal drug formulations mayincorporate components such as polyethylene glycol (PEG) (see Klibanovet al. (1990) FEBS Lett. 268: 235-7; Mayuryama et al. (1992) Biochim.Biophys. Acta 1128: 44-49; Allen et al. (1991) Biochim. Biophys. Acta1066: 29-36). PEG conjugation to immunoliposomes has been shown toprolong liposome circulation in blood, as well as to enhance thetherapeutic efficacy of liposomal drugs (Daemen et al. (1997) J. ControlRel. 44: 1-9; Storm et al. (1998) Clin. Cancer Res. 4: 111-115; Vaage etal. (1997) Br. J. Cancer 75: 482-6; Gabizon et al. (1994) Cancer Res.54: 987-92). Long-circulating immunoliposomes can be classified into twotypes: those with antibodies coupled to a lipid head growth (Maruyama etal. (1990) J. Pharm. Sci. 74: 978-84); and those with antibodies coupledto the distal end of PEG (Maruyama et al. (1997) Adv. Drug Del. Rev. 24:235-42). In certain instances, it way be advantageous to place thetumor-specific antibodies at the distal end of the PEG polymer to obtainefficient target binding by avoiding steric hindrance from the PEGchains. This type of immunoliposome formulation has been usedsuccessfully for in vivo targeting to the lungs (Maruyama et al. (1995)Biochim. Biophys. Acta 1234: 74-80; brain (Huwyler et al. (1996) Proc.Natl. Acad. Sci. USA 93: 14164-69); and tumors (Allen et al. (1995)Biochem. Soc. Transact. 23: 1073-79).

Effective delivery of drugs by immunoliposome formulations is generallyenhanced by active uptake of the bound immunoliposome throughendocytosis. Human scFv antibodies can be selected for optimizedinternalization into tumor cells from a phage display library to ensureoptimal targeting and delivery of the immunoliposomes into which theyare incorporated (see Poul et al. (2000) J. Mol. Biol. 301: 1149-61;Schier et al., (1996) J. Mol. Biol. 263: 551-67).

Another strategy related to antibody-mediated tumor targeting isantibody-directed enzyme pro-drug (ADEPT), which is a two steptherapeutic approach designed to generate a high concentration ofanticancer drugs in proximity to tumor cell membranes (Springer et al.(1996) Adv. Drug Deliv. Rev. 22: 351-64). Using this strategy, anenzyme-antibody conjugate that preferentially binds to a giventumor-associated antigen is administered first, followed by injection ofa nontoxic prod-drug, which becomes activated by the action of thetargeted enzyme. An improved ADEPT using immunoliposomes as a targetedcarrier for the pro-drug-activating enzymes instead of anenzyme-antibody conjugate has been developed and tested (Storm et al.(1997) Adv. Deliv. Rev. 24: 225-31; Vingerhoeds et al. (1993) FEBS Lett.336: 485-90).

Therapies

The invention provides for treatment or prevention of cancer, including,but not limited to, neoplasms, tumors, metastases, or any disease ordisorder characterized by uncontrolled cell growth, and particularlymultidrug resistant forms thereof by the administration oftherapeutically or prophylactically effective amounts of anti-vimentinantibodies or nucleic acid molecules encoding said antibodies. Examplesof types of cancer and proliferative disorders to be treated with thevimentin-targeted therapeutics of the invention include, but are notlimited to, leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic,monocytic, erythroleukemia, chronic myelocytic (granulocytic) leukemia,and chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's disease andnon-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma,Ewing's tumor, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, renal cell carcinoma, hepatoma, Wilms' tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma,dysplasia and hyperplasia. In a particular embodiment, therapeuticcompounds of the invention are administered to men with prostate cancer(e.g., prostatitis, benign prostatic hypertrophy, benign prostatichyperplasia (BPH), prostatic paraganglioma, prostate adenocarcinoma,prostatic intraepithelial neoplasia, prostato-rectal fistulas, andatypical prostatic stromal lesions). The treatment and/or prevention ofcancer includes, but is not limited to, alleviating symptoms associatedwith cancer, the inhibition of the progression of cancer, the promotionof the regression of cancer, and the promotion of the immune response.In one embodiment, commercially available or naturally occurringanti-vimentin antibodies, functionally active fragments or derivativesthereof are used in the present invention.

The vimentin therapeutics may be administered alone or in combinationwith other types of cancer treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Examples of anti-tumor agents include, but are not limited to,cisplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors(e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine,oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal,and taxol. In one embodiment, one or more anti-vimentin antibodies areadministered to an animal, preferably a mammal and most preferably ahuman, after surgical resection of cancer. In another embodiment, one ormore anti-vimentin antibodies are administered to an animal, preferablya mammal and most preferably a human, in conjugation with chemotherapyor radiotherapy. In another embodiment, one or more anti-vimentinantibodies are administered to an animal, preferably a mammal and mostpreferably a human, for the prevention or treatment of cancer prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of plasma to theanimal.

The anti-vimentin antibodies, and other vimentin-targeted therapeuticsdescribed herein, may be administered to an animal, preferably a mammaland most preferably a human, for the prevention or treatment of cancerprior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 weekbefore), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours,2 days, or 1 week after), or concomitantly with the administration ofIgG antibodies, IgM antibodies and/or one or more complement componentsto the animal. In another preferred embodiment, one or moreanti-vimentin antibodies are administered to an animal, preferably amammal and most preferably a human, prior to (e.g., 1 minute, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after),or concomitantly with the administration of antibodies immunospecificfor one or more cancer cell antigens. In yet another preferredembodiment, one or more anti-vimentin antibodies are administered to ananimal, preferably a mammal and most preferably a human, for theprevention or treatment of cancer prior to (e.g., 1 minute, 15 minutes,30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), orconcomitantly with the administration of antibodies currently used forthe treatment of cancer. Examples of such antibodies include, but arenot limited to, Herceptin, Retuxan, OvaRex, Panorex, BEC2, IMC-C225,Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart I D10, and Oncolym.

The invention further provides methods for the treatment or preventionof viral and other pathogen infections in an animal, preferably a mammaland most preferably a human, said methods comprising the administrationof a therapeutically or prophylactically effective amount ofanti-vimentin antibodies or nucleic acid molecules encoding saidantibodies or other vimentin-targeted therapeutics described herein.Examples of viral infections which can be treated or prevented inaccordance with this invention include, but are limited to, viralinfections caused by retroviruses (e.g., human T-cell lymphotrophicvirus (HTLV) types I and II and human immunodeficiency virus (HIV)),herpes viruses (e.g., herpes simplex virus (HSV) types I and II,Epstein-Barr virus and cytomegalovirus), arenaviruses (e.g., lassa fevervirus), paramyxoviruses (e.g., morbillivirus virus, human respiratorysyncytial virus, and pneumovirus), adenoviruses, bunyaviruses (e.g.,hantavirus), cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses(e.g., hepatitis C virus (HCV), yellow fever virus, and Japaneseencephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)),orthomyoviruses (e.g., Sendai virus and influenza viruses A, B and C),papovaviruses (e.g., papillomavirues), picornaviruses (e.g.,rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses,reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus), andrhabdoviruses (e.g., rabies virus). The treatment and/or prevention of aviral infection includes, but is not limited to, alleviating symptomsassociated with said infection, the inhibition or suppression of viralreplication, and the enhancement of the immune response.

The vimentin-targeted therapeutics described herein may be administeredalone or in combination with other types of anti-viral or otheranti-pathogen agents. Examples of anti-viral agents include, but are notlimited to: cytokines (e.g., IFN-.alpha., IFN-.beta., and IFN-.gamma);inhibitors of reverse transcriptase (e.g., AZT, 3TC, D4T, ddC, ddI, d4T,3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir, and otherdideoxynucleosides or dideoxyfluoronucleosides); inhibitors of viralmRNA capping, such as ribavirin; inhibitors of proteases such HIVprotease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir,and saquinavir); amphotericin B; castanospermine as an inhibitor ofglycoprotein processing; inhibitors of neuraminidase such as influenzavirus neuraminidase inhibitors (e.g., zanamivir and oseltamivir);topoisomerase I inhibitors (e.g., camptothecins and analogs thereof);amantadine and rimantadine. For example, one or more anti-vimentinantibodies-drug conjugates are administered to an animal, preferably amammal and most preferably a human, for the prevention or treatment of aviral infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours,2 days, or 1 week before subsequent to (e.g., 1 minute, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12hours, 24 hours, 2 days, or 1 week after), or concomitantly with theadministration of plasma to the animal.

In other examples, one or more vimentin-targeted therapeutics areadministered to an animal, preferably a mammal and most preferably ahuman, for the prevention or treatment of a viral infection prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of IgGantibodies, IgM antibodies and/or one or more complement components tothe animal. In another preferred embodiment, anti-vimentin antibodiesare administered to an animal, preferably a mammal and most preferably ahuman, for the prevention or treatment of a viral infection prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of antibodiesimmunospecific for one or more viral antigens. Example of antibodiesimmunospecific for viral antigens include, but are not limited to,Synagis®, PRO542, Ostavir, and Protovir.

The invention further provides methods for the treatment or preventionof microbial infections in an animal, preferably a mammal and mostpreferably a human, said methods comprising the administration of atherapeutically or prophylactically effective amount ofanti-vimentin-targeted therapeutics. Examples of microbial infectionswhich can be treated or prevented in accordance with this inventioninclude, but are not limited to, yeast infections, fungal infections,protozoan infections and bacterial infections. Bacteria which causemicrobial infections include, but are not limited to, Streptococcuspyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseriameningitidis, Corynebacterium diphtheriae, Clostridium botulinum,Clostridium perfringens, Clostridium tetani, Haemophilus influenzae,Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis,Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonasaeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni,Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersiniaenterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigelladysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium,Treponema pallidum, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii,Francisella tularensis, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp., and Helicobacter pylori. The treatmentand/or prevention of a microbial infection includes, but is not limitedto, alleviating symptoms associated with said infection, the inhibitionor suppression of replication, and the enhancement of the immuneresponse.

Vimentin-targeted therapeutics may be administered alone or incombination with other types of anti-microbial agents. Examples ofanti-microbial agents include, but are not limited to: antibiotics suchas penicillin, amoxicillin, ampicillin, carbenicillin, ticarcillin,piperacillin, cepalospolin, vancomycin, tetracycline, erythromycin,amphotericin B, nystatin, metroidazole, ketoconazole, and pentamidine.In one embodiment, a vimentin-targeted therapeutic is administered to ananimal, preferably a mammal and most preferably a human, for theprevention or treatment of a microbial infection prior to (e.g., 1minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after) or concomitantly with the administration of plasma to theanimal.

In certain instances, one or more vimentin-targeted therapeutics areadministered to an animal, preferably a mammal and most preferably ahuman, for the prevention or treatment of a microbial infection prior to(e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before),subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or1 week after), or concomitantly with the administration of IgGantibodies, IgM antibodies and/or one or more complement components tothe animal. In other instances, one or more vimentin-targetedtherapeutics are administered to an animal, preferably a mammal and mostpreferably a human, for the prevention or treatment of a microbialinfection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes,1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days,or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12hours, 24 hours, 2 days, or 1 week after), or concomitantly with theadministration of antibodies immunospecific for one or more microbialantigens. Example of antibodies immunospecific for microbial antigensinclude, but are not limited to, antibodies immunospecific for LPS andcapsular polysaccharide 5/8. In certain embodiments, animals withincreased risk of a viral or bacterial infection are administered acomposition of the invention. Examples of such animals include, but arenot limited to, human burn patients, infants, immunocompromised orimmunodeficient humans, and the elderly.

4.6 Kits

The invention further provides kits for use in diagnostics orprognostic, as well as therapeutic, methods for neoplasias and multidrugresistant neoplasias. The diagnostic kits are useful, for example, fordetecting cell surface vimentin-expressing neoplasias and for monitoringthe occurrence of multidrug resistant cells in a patient sample or insitu in a patient. For example, during the course of patientchemotherapeutic treatment, monitoring of cell surface vimentin, andother MDR-associated markers described herein, provides valuableinformation regarding the efficacy of the treatment and for avoiding thedevelopment of multidrug resistance. For example, the kit can comprise alabeled compound or agent capable of detecting cell surface vimentinprotein in a biological sample; as well as means for determining theamount of cell surface vimentin in the sample; and means for comparingthe amount of vimentin in the sample with a standard (e.g., normalnon-neoplastic cells or non-MDR neoplastic cells). The compound or agentcan be packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect cell surface vimentin protein,as well as other MDR-associated markers. Such a kit can comprise, e.g.,one or more antibodies capable of binding specifically to at least aportion of a cell surface vimentin protein.

4.7 Vimentin Vaccines

Immunological compositions, including vaccines, and other pharmaceuticalcompositions containing the vimentin protein, or portions thereof, areincluded within the scope of the present invention. One or more of thevimentin proteins, or active or antigenic fragments thereof, or fusionproteins thereof can be formulated and packaged, alone or in combinationwith other antigens, using methods and materials known to those skilledin the art for vaccines. The immunological response may be usedtherapeutically or prophylactically and may provide antibody immunity orcellular immunity, such as that produced by T lymphocytes.

To enhance immunogenicity, the proteins may be conjugated to a carriermolecule. Suitable immunogenic carriers include proteins, polypeptidesor peptides such as albumin, hemocyanin, thyroglobulin and derivativesthereof, particularly bovine serum albumin (BSA) and keyhole limpethemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solidphases. Other protein derived or non-protein derived substances areknown to those skilled in the art. An immunogenic carrier typically hasa molecular mass of at least 1,000 Daltons, preferably greater than10,000 Daltons. Carrier molecules often contain a reactive group tofacilitate covalent conjugation to the hapten. The carboxylic acid groupor amine group of amino acids or the sugar groups of glycoproteins areoften used in this manner. Carriers lacking such groups can often bereacted with an appropriate chemical to produce them. Preferably, animmune response is produced when the immunogen is injected into animalssuch as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, andother animals, most preferably mice and rabbits. Alternatively, amultiple antigenic peptide comprising multiple copies of the protein orpolypeptide, or an antigenically or immunologically equivalentpolypeptide may be sufficiently antigenic to improve immunogenicitywithout the use of a carrier.

The vimentin protein or portions thereof, such as consensus or variablesequence amino acid motifs, or combination of proteins may beadministered with an adjuvant in an amount effective to enhance theimmunogenic response against the conjugate. One adjuvant widely used inhumans has been alum (aluminum phosphate or aluminum hydroxide). Saponinand its purified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications are alsoavailable. Chemically defined preparations such as muramyl dipeptide,monophosphoryl lipid A, phospholipid conjugates such as those describedby Goodman-Snitkoff et al. (1991) J. Immunol. 147:410-415 andincorporated by reference herein, encapsulation of the conjugate withina proteoliposome as described by Miller et al. (1992) J. Exp. Med.176:1739-1744 and incorporated by reference herein, and encapsulation ofthe protein in lipid vesicles such as Novasome™ lipid vesicles (MicroVescular Systems, Inc., Nashua, N.H.) may also be useful.

The invention includes the vimentin polypeptide fragments, orsubsequences of the intact vimentin polypeptide shown in FIG. 12A (SEQID NO. 1). Such vimentin polypeptide subsequences, or a correspondingnucleic acid sequence that encodes them in the case of DNA vaccines, arepreferably selected so as to be highly immunogenic. The principles ofantigenicity for the purpose of producing anti-vimentin vaccines applyalso to the use of vimentin polypeptide sequences for use as immunogensfor generating anti-vimentin polyclonal and monoclonal antibodies foruse in the vimentin-based diagnostics and therapeutics described herein.

Computer assisted algorithms for predicting polypeptide subsequenceantigenicity are widely available. For example “Antigenic” looks forpotential antigenic regions using the method of Kolaskar (see Kolaskarand Tongaonkar (1990) FEBS Letters 276:172-174 “A semi-empirical methodfor prediction of antigenic determinants on protein antigens”). In theirinitial study, Kolaskar and Tongaonkar experimentally tested 169antigenic. The 156 which have less than 20 amino acids per determinantwere selected (total 2066 residues). f(Ag) was calculated as thefrequency of occurrence of each residue in antigenic determinants[f(Ag)=Epitope_occurrence/2066]. The Hydrophilicity, Accessibility andFlexibility values are from Parker, et al. (see Parker, et al. (1986)Biochemistry 25:5425-5432). In a given protein, the average for each7-mer is calculated, and values are assigned to the central residue ofthe 7-mer. A residue is considered to be on the surface if any of the7-mer values was above the average for the protein. These results wereused to obtain f(s) as the frequency of occurrence of amino acids at thesurface. The prediction algorithm includes the following steps:calculate the average propensity for each overlapping 7-mer and assignthe result to the central residue (i+3) of the 7-mer; calculate theaverage for the whole protein; if the average for the whole protein isabove 1.0 then all residues having above 1.0 are potentially antigenic;if the average for the whole protein is below 1.0 then all residueshaving above the average for the whole protein (note: the original paperhas a mangled formula here) are potentially antigenic; find 6-mers whereall residues are selected by step 3.

Another method for determining antigenicity of a polypeptide subsequenceis the algorithm of Hopp and Woods ((1981) Proc. Natl. Acad. Sci. 86:152-6). There are publicly available web sites for Hopp and Woodsalgorithm analysis of a user-input polypeptide sequence and convenientgraphical output of the resulting analysis (see, e.g.,http://hometown.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/7/). Usingthis algorithm to analyze the full-length human vimentin sequence shownin FIG. 12A, several suitable sequence having a high Hopp and Woodsantigenic index of an adequate length for immunogenicity were revealed.These include vimentin amino acid residues: 45-60 (i.e.,RPSTSRSLYASSPGGV); 295-315 (i.e., FADLSEAANRNNDALRQAKQE) and 330-345(i.e., VDALKGTNESLERQMR).

In addition, the present invention provides a composition comprising thevimentin protein or polypeptide fragment of the invention in combinationwith a suitable adjuvant. Such a composition can be in apharmaceutically acceptable carrier, as described herein. As usedherein, “adjuvant” or “suitable adjuvant” describes a substance capableof being combined with the vimentin protein or polypeptide to enhance animmune response in a subject without deleterious effect on the subject.A suitable adjuvant can be, but is not limited to, for example, animmunostimulatory cytokine, SYNTEX adjuvant formulation 1 (SAF-1)composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.Other suitable adjuvants are well known in the art and include QS-21,Freund's adjuvant (complete and incomplete), alum, aluminum phosphate,aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion. The adjuvant, such as an immunostimulatory cytokine can beadministered before the administration of the vimentin protein orvimentin-encoding nucleic acid, concurrent with the administration ofthe vimentin protein or vimentin-encoding nucleic acid or up to fivedays after the administration of the vimentin protein orvimentin-encoding nucleic acid to a subject. QS-21, similarly to alum,complete Freund's adjuvant, SAF, etc., can be administered within hoursof administration of the fusion protein.

The invention may also utilize combinations of adjuvants, such asimmunostimulatory cytokines co-administered to the subject before, afteror concurrent with the administration of the vimentin protein orvimentin-encoding nucleic acid. For example, combinations of adjuvants,such as immunostimulatory cytokines, can consist of two or more ofimmunostimulatory cytokines of this invention, such as GM/CSF,interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumornecrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L,B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules. Theeffectiveness of an adjuvant or combination of adjuvants may bedetermined by measuring the immune response directed against thevimentin polypeptide with and without the adjuvant or combination ofadjuvants, using standard procedures, as described herein.

Furthermore, the present invention provides a composition comprising thevimentin protein or vimentin-encoding nucleic acid and an adjuvant, suchas an immunostimulatory cytokine or a nucleic acid encoding an adjuvant,such as an immunostimulatory cytokine. Such a composition can be in apharmaceutically acceptable carrier, as described herein. Theimmunostimulatory cytokine used in this invention can be, but is notlimited to, GM/CSF, interleukin-2, interleukin-12, interferon-gamma,interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoieticfactor flt3L, CD40L, B7.1 con-stimulatory molecules and B7.2co-stimulatory molecules.

The term “vaccine” as used herein includes DNA vaccines in which thenucleic acid molecule encoding vimentin or antigenic portions thereof,such as any consensus or variable sequence amino acid motif, in apharmaceutical composition is administered to a patient. For geneticimmunization, suitable delivery methods known to those skilled in theart include direct injection of plasmid DNA into muscles (Wolff et al.(1992) Hum. Mol. Genet. 1:363), delivery of DNA complexed with specificprotein carriers (Wu et al. (1989) J. Biol. Chem. 264:16985,coprecipitation of DNA with calcium phosphate (Benvenisty and Reshef(1986) Proc. Natl. Acad. Sci. 83:9551), encapsulation of DNA inliposomes (Kaneda et al. (1989) Science 243:375), particle bombardment(Tang et al., (1992) Nature 356:152, and Eisenbraun et al. (1993) DNACell Biol. 12:791), and in vivo infection using cloned retroviralvectors (Seeger et al. (1984) Proc. Natl. Acad. Sci. 81:5849).

In another embodiment, the invention is a polynucleotide which comprisescontiguous nucleic acid sequences capable of being expressed to producea vimentin or immunostimulant gene product upon introduction of saidpolynucleotide into eukaryotic tissues in vivo. The encoded gene productpreferably either acts as an immunostimulant or as an antigen capable ofgenerating an immune response. Thus, the nucleic acid sequences in thisembodiment encode an immunogenic epitope, and optionally a cytokine or aT-cell costimulatory element, such as a member of the B7 family ofproteins.

Advantages to immunization with a gene rather than its gene productinclude the following. First, is the relative simplicity with whichnative or nearly native antigen can be presented to the immune system.Mammalian proteins expressed recombinantly in bacteria, yeast, or evenmammalian cells often require extensive treatment to ensure appropriateantigenicity. A second advantage of DNA immunization is the potentialfor the immunogen to enter the MHC class I pathway and evoke a cytotoxicT cell response Immunization of mice with DNA encoding the influenza Anucleoprotein (NP) elicited a CD8⁺ response to NP that protected miceagainst challenge with heterologous strains of flu. (Montgomery, D. L.et al. (1997) Cell Mol Biol 43(3):285-92; and Ulmer, J. et al. (1997)Vaccine 15(8):792-794). Cell-mediated immunity is important incontrolling infection. Since DNA immunization can evoke both humoral andcell-mediated immune responses, its greatest advantage may be that itprovides a relatively simple method to survey a large number of vimentingenes and gene fragments for their vaccine potential.

The invention also includes known methods of preparing and using tumorantigen vaccines for use in treating or preventing cancers. For example,U.S. Pat. No. 6,562,347 which teaches the use of a fusion polypeptideincluding a chemokine and a tumor antigen which is administered aseither a protein or nucleic acid vaccine to elicit an immune responseeffective in treating or preventing cancer. Chemokines are a group ofusually small secreted proteins (7-15 kDa) induced by inflammatorystimuli and are involved in orchestrating the selective migration,diapedesis and activation of blood-born leukocytes that mediate theinflammatory response (see Wallack (1993) Annals New York Academy ofSciences 178). Chemokines mediate their function through interactionwith specific cell surface receptor proteins (23). At least fourchemokine subfamilies have been identified as defined by a cysteinesignature motif, termed CC, CXC, C and CX₃C, where C is a cysteine and Xis any amino acid residue. Structural studies have revealed that atleast both CXC and CC chemokines share very similar tertiary structure(monomer), but different quaternary structure (dimer). For the mostpart, conformational differences are localized to sections of loop orthe N-terminus. In the instant invention, for example, a human vimentinpolypeptide sequence (such as that shown in FIG. 12A), or polypeptidefragment thereof, and a chemokine sequence are fused together and usedin an immunizing vaccine. The chemokine portion of the fusion can be ahuman monocyte chemotactic protein-3, a human macrophage-derivedchemokine or a human SDF-1 chemokine. The vimentin portion of the fusionis, preferably, a portion shown in routine screening to have a strongantigenic potential.

4.8 Pharmaceutical Formulations and Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject having a neoplastic disease. Subjects atrisk for such a disease can be identified by a diagnostic or prognosticassay, e.g., as described herein. Administration of a prophylactic agentcan occur prior to the manifestation of symptoms characteristic of theneoplasm, such that development of the neoplasm is prevented or,alternatively, delayed in its progression. In general, the prophylacticor therapeutic methods comprise administering to the subject aneffective amount of a compound which comprises a vimentin targetingcomponent that is capable of binding to cell surface vimentin present onneoplastic, and particularly multidrug resistant neoplastic, cells andwhich compound is linked to a therapeutic component.

Examples of vimentin targeting components include monoclonalanti-vimentin antibodies and fragments thereof. Examples of suitabletherapeutic components include traditional chemotherapeutic agents suchas Actinomycin, Adriamycin, Altretamine, Asparaginase, Bleomycin,Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil,Cisplatin, Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine,Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide,Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin,Ifosfamide, Imatinib, Irinotecan, Lomustine, Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,Vinblastine, Vincristine, and Vinorelbine. Other examples of suitabletherapeutic components include immunotoxins such as Pseudomonasexotoxin, a diphtheria toxin, a plant ricin toxin, a plant abrin toxin,a plant saporin toxin, a plant gelonin toxin, and pokeweed antiviralprotein. Such immunotoxins are targeted to the vimentin expressingneoplastic, or multidrug resistant neoplastic, cell by the vimentintargeting component of the therapeutic compound and, upon binding ofcell surface vimentin and uptake into the cell, function to kill orblock the growth of the neoplastic cell.

4.8.1 Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (The Dose Lethal To 50% Of ThePopulation) And The ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic induces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

4.8.2 Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsulation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remrnington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable delivery systems includemicrospheres which offer the possibility of local noninvasive deliveryof drugs over an extended period of time. This technology utilizesmicrospheres of precapillary size which can be injected via a coronarycatheter into any selected part of the e.g. heart or other organswithout causing inflammation or ischemia. The administered therapeuticis slowly released from these microspheres and taken up by surroundingtissue cells (e.g. endothelial cells).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

In clinical settings, a therapeutic and gene delivery system for thevimentin-targeted therapeutic can be introduced into a patient by any ofa number of methods, each of which is familiar in the art. For instance,a pharmaceutical preparation of the vimentin-targeted therapeutic can beintroduced systemically, e.g., by intravenous injection.

The pharmaceutical preparation of the vimentin-targeted therapeuticcompound of the invention can consist essentially of the compound in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle or compound is imbedded.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

5. EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference. Nucleotide and aminoacid sequences deposited in public databases as referred to herein arealso hereby incorporated by reference. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 5.1 Overexpression of a 53 kDa Protein in MDR SolidTumor/Cancer Cells

Studies were performed to determine what proteins, if any, weredifferentially expressed in multidrug resistant tumor cell lines ascompared to their drug-sensitive counterparts. The seven different celllines used in the Examples are described in Table I below.

TABLE I Multidrug resistant cell line derived from a clone Cancer celltissue Drug-sensitive of the “parent” drug- type “parent” cell linesensitive cell line* Source of cells Promyelocytic HL60 HL60/AR AmericanTissue leukemia Culture Collection (ATCC, Manassas, VA) & AureliumBioPharma Promyelocytic NB4 NB4/VLB Deutsche Sammlung leukemia NB4/DOXvon Midroorganismen und Zellkulturen GmbH (DSMZ, Germany) & AureliumBioPharma T lymphoblastoid CEM CEM/VLB ATCC, Dr. William CEM/DOX Beckand Aurelium BioPharma T lymphoblastoid HSB2 HSB2/VLB ATCC and AureliumHSB2/DOX BioPharma T lymphoblastoid Molt4 Molt4/DOX ATCC and AureliumMolt4/VLB BioPharma Breast epithelial MCF-7 MCF-7/AR ATCC and AureliumMDA/MITO BioPharma Breast epithelial MDA MDA/AR ATCC and AureliumMDA/MITO BioPharma Ovarian SKOV-3 SKOV-3/T320 ATCC and Aurelium 20082008/T320 BioPharma *MDR cells lines are named systematically using theparent cell line followed by a forward slash and abbreviation for thename of the drug used in selecting resistance in the parent cell line.Drug abbreviations in this table include: AR (adriamycin); VLB(vinblastine); DOX (doxorubicin); MITO (mitomycin); and T320 (taxol).

Suspension cells were grown in RPMI or α-MEM medium, containing 10% to15% fetal calf serum (commercially available from Hyclone Inc., Logan,Utah). The cells were grown in the absence of antibiotics at 37° C. inhumid atmosphere of 5% CO₂ and 95% air, and passaged when cultures were1×10⁶ cells/ml. Multidrug resistant cells (HL60/AR, NB4/VLB, NB4/DOX,CEM/VLB, CEM/DOX, HSB2/VLB, HSB2/DOX, Molt4/VLB Molt4/DOX) (AureliumBiopharma Inc., Montreal (Quebec), Canada) were grown continuously withappropriate concentrations of cytotoxic drugs. Similarly, adherent cellswere grown in α-MEM medium (MCF-7) or DMEM (MDA), containing 10% fetalcalf serum. Multidrug resistant cells (MCF-7/AR, MDA/AR and MDA/MITO)were grown continuously with appropriate concentrations of cytotoxicdrugs. All cell lines were examined for and determined to be free ofmycoplasma contamination using a PCR-based mycoplasma detection kitaccording to manufacturer's instructions commercially available (e.g.,from Stratagene Inc., San Diego, Calif.). All multidrug resistant celllines were routinely tested for multidrug resistance using a panel ofdifferent drugs representing different classes of drugs. The MDR cellsalso expressed other MDR markers on their cell surface.

Different types of extracts were prepared from each cell type. Cellswere concentrated and lysed according to standard procedures to obtaintotal cell extracts from the cells (e.g., Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons Inc., New York City,N.Y. 1993). Alternatively, cells were first surface biotinylated andthen lysed to obtain biotinylated total cell extracts (as shown inExamples below). To do these studies, intact drug sensitive (CEM, HL60and MCF-7) and multidrug resistant cells (CEM/VLB, HL60/AR and MCF-7/AR)were biotinylated with a membrane impermeable biotinylating agent,Sulfo-NHS-LC-LC-Biotin (Pierce #21338). To do this, cells werebiotinylated by washing 3× with 50 ml PBS, pH 8. Next,Sulfo-NHS-LC-LC-Biotin, which is a membrane impermeable reagent, wasprepared at 0.1-0.5 mg/ml and added to cells. The incubation withSulfo-NHS-LC-LC-Biotin was allowed to continue for 1 hour at 4° C. withrotation. The reaction was stopped by washing cells one time with 50 mlPBS pH 8, containing 10 mM glycine and several times with 50 ml PBS,without glycine. Cells were then lysed in 200 μl of buffer A (1% SDS and0.05 M Tris/HCl, pH 7.4), containing proteases inhibitors (Proteasesinhibitors: 1 μg/ml pepstatin, 1 μg/ml leupeptin; 1 μg/ml benzamidine;0.2 mM PMSF) and incubated 5 minutes on ice. The cell lysate was thensonicated with a Vibracell sonicator amplitude 40 setting #25 for 3×10seconds with 1 minute on ice between shots. The sonicated cell lysatewas mixed with 800 μl of buffer B (1.25% Triton-X100, 0.05 M Tris/HCl,pH 7.4, 190 mM NaCl), containing proteases inhibitors and incubated 5minutes on ice. The cell lysate was next centrifuged at 14,000 rpm in anEppendorf microfuge for 5 minutes. The supernatant was removed, and itsprotein concentration was determined using the DC protein assay kit fromBIORAD according to manufacturer's instructions (BioRad Laboratories,Hercules, Calif.) (see also Lowry et al., J. Biol. Chem. 193: 265-275,1951).

The use of the sulfo-LC-LC-biotinylating agent ensured the modificationof the c amino group on the lysine side chain in proteins exposed on thecell surface; conversely, intracellular proteins were not expected to bebiotinylated since this sulfo-biotin cannot cross the cell membrane ofintact cells.

In addition, plasma membrane preparations were prepared from surfacebiotinylated or nonbiotinylated cells of each type. To do this, 3×10⁹cells (of each cell type) were suspended in 12.5 ml of hypotonic buffer1 (10 mM NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl pH 7.4) and incubated for 10min on ice. The cells were then homogenized in a Dounce glasshomogenizer type B (15 ml). The degree of cell lysis was determined byexamining cells under the microscope. Approximately 40 strokes wererequired to break about 85% of the cells. Immediately afterhomogenization, half volume (6.25 ml) of 2.5× buffer II (Buffer 1×: 210mM mannitol, 70 mM sucrose, 5 mM Tris-HCl pH 7.5, 1 mM EDTA pH 7.5) wasadded to the cell homogenate and mixed. The homogenate was spun at1300×g (3300 rpm) for 5 min in a Sorvall centrifuge using SS34 rotor(brake off). The pellet containing the nuclei fraction was separatedfrom the supernatant containing cell membranes and organelles. Thepost-nuclei supernatant was spun or centrifuged again at 17000×g (11900rpm) 15 min in a Sorvall centrifuge using the SS34 rotor (brake off).The mitochondrial-enriched pellet was separated from the membraneenriched supernatant fraction (post-mitochondrial fraction). The lattersupernatant was centrifuged for 2 hours at 100,000×g in the SorvallUltracentrifuge using the AH-629 rotor and PA UltraClear tubes fromBeckman #cat 344058 at 4° C. The cytosolic enriched supernatant wascarefully removed and the membrane enriched membrane pellet wasresuspended in 300 μl of buffer 1 above and mixed well using a 27 gaugeneedle.

The cell membranes were further enriched by resolving the last membranepellet on a discontinuous sucrose gradient (16%, 31%, 45%, 60% w/vsucrose/buffer 1). Briefly, equal volume (about 350 μl) of 32% w/v ofsucrose in buffer 1 (16% w/v final) was added to the resuspended pelletof enriched membrane material following 100,000×g centrifugation step.The sucrose gradient was prepared with 6.9 ml 60% at the bottom of thetube followed by 9.9 ml of 45% sucrose, 13.9 ml 31% sucrose and 6.9 mlof 16% sucrose. A 16% sucrose containing crude plasma membranes wasslowly poured on the top of the gradient and the sample spun for 18hours at 100,000×g at 4° C. in the Sorvall ultracentrifuge using theAH-629 rotor and PA UltraClear tubes from Beckman #cat 344058. Theinterphase between the 16% and 31% sucrose, containing a highly enrichedcell membrane was collected and washed once by centrifugation withbuffer 1. Following a 100,000×g centrifugation in the ultracentrifugethe highly enriched cell membrane pellet was resuspended in anappropriate volume (about 50 μl) of buffer 1 and stored at −80° C.

Equivalent amounts of protein from surface biotinylated total cellextracts (containing biotinylated and non-biotinylated proteins) andplasma membrane preparations from each of the cell types (HL60, HL60/AR,NB4, NB4/DOX, NB4/VLB, CEM, CEM/VLB, CEM/DOX, Molt4, Molt4/AR,Molt4/VLB, HSB2, HSB2/VLB, HSB2/DOX, MCF-7, MCF-7/AR, MDA, MDA/AR,MDA/MITO) were analyzed by two dimensional (2-D) gel electrophoresis andvisualized by either silver staining or immunoblotting with anti-VIMantibody or streptavidin-HPR conjugate (streptavidin binds biotin). Thisallowed resolution of protein samples according to differences in theirisoelectric points in the first dimension and molecular masses in thesecond dimension. For the first dimension, isoelectric focusing wasachieved using 13 cm immobilized pH gradient strips (Amersham PharmaciaBiotech, Piscataway, N.J.). Briefly, 13 cm strips were rehydrated in aceramic strip holder in 250 μl rehydration buffer containing the proteinsamples (0.5 mg-2 mg proteins) for 15 hours at 30 volts. Electrode padswere then placed over each electrode and the proteins separated on anIPGphor unit using the following program:

13 cm strips (pH 4-7):

-   -   −500V for 500 Vh    -   −1000V for 1000 Vh    -   −8000V for 16000 Vh        Strips were then slightly rinsed with water and equilibrated in        1% DTT in equilibration buffer for 15 min, followed by 4%        iodoacetamide in equilibration buffer for 15 minutes.

For the second dimension, the above isoelectric strips were subject tosodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)using an 8% or 10% gel, according to the method of Laemmli (LaemmliU.K., Nature 227:680-685, 1970). Molecular weight markers were loadedonto a 2×3 mm filter paper and placed at one end of the strip. The stripand molecular weight marker filter were then sealed onto thepolyacrylamide gel with a 0.5% agarose solution in running buffer.Proteins were slowly transferred from the strip to the gel at 30 V atroom temperature for an hour and the separation was carried out at 4-8°C. for 17-18 hours at 70 V or 75 V for 8% or 10% gels respectively.

The gels were next stained with silver stain, and photographed. (Note,that at this point, the gel-resolved proteins can also be transferredonto Hybond C nitrocellulose membrane and then immunoblotted withantibody or streptavidin-HRP, as described below in the Examples).

An about 53 kDa protein (with a pI of about 5) was identified in silverstained 2D-gels of total cell extracts of MCF-7 and MCF-7/AR cells. The53 kDa protein was highly overexpressed in the total cell extracts ofmultidrug resistant MCF-7/AR cells compared to drug sensitive MCF-7cells (FIGS. 1(A) and (B)).

Example 2 5.2 Identification of the 53 kDa Protein of MCF-7/AR BreastCancer Cells as Vimentin

To discover the identity of the 53 kDa protein of breast MCF-7 andMCF-7/AR cells, 2-D gels were loaded with MCF-7 or MCF-7/AR total cellsextract (2×750 μg, pI 4 to 7, 10% gel), silver-stained, and the 53 kDaspots were excised. The spots were next processed using optimizedprocedures for staining/destaining of gels, trypsin digestion, peptideextraction and peptide purification. Briefly, gels were stained withSilverQuest silver stain (Invitrogen, Carlsbad, Calif.) using the recipegiven by the manufacturer. The protein spots of interest were excisedwith an acid washed razor blade and cut into small pieces on a clean(acid washed) glass plate and transferred to a 200 μl PCR tube (MeOHtreated). The gel pieces were mixed with 50 μl destainer A and 50 μldestainer B (provided with SilverQuest kit) (or 100 μl of the destainerspremix prepared fresh), and incubated for 15 min at room temperaturewithout agitation. The wash was removed using a capillary tip. Water wasadded to the gel pieces, mixed and incubated 10 min at room temperature.The latter step was repeated three times. The gel pieces were thendehydrated in 100 μl 100% methanol for 5 minutes at room temperature,followed by re-hydration in 30% methanol/water for 5 min. Gel pieceswere then washed twice in water for ten minutes and twice in 25 mM Ambic(ammonium bicarbonate)/30% acetonitrile (for 5 minutes) followed by agel drying step. Tryptic digestion of the destained and washed gelpieces was performed by adding about 1 volume of trypsin solution (130ng of enzyme in 25 mM ammonium bicarbonate, 5 mM CaCl₂) to 1 volume ofgel pieces and the samples were left on ice for 45 minutes. Thedigestion was allowed to proceed for 15-16 hr at 37° C. Digestedpeptides were extracted with acetonitrile for 15 minutes at roomtemperature with shaking. The gel pieces/solvent were sonicated 5minutes and re-extracted with 25 mM Ambic:50% acetonitrile withoutsonication. Digested peptides were further extracted with 5% formicacid:50% acetonitrile:45% water freshly prepared at room temperaturewith shaking for 15 minutes. The mix was completed with one volume ofacetonitrile and the gel pieces/solvent were sonicated 5 minutes andre-extracted the same way without sonication. The collected material wascombined and dried. The extracted peptides were re-suspended in 5%methanol with 0.2% trifluoroacetic acid (or 0.5% formic acid) thenloaded on an equilibrated C18 bed (Ziptip from Millipore). The loadedZiptip was washed with 5% acetonitrile containing 0.2% TFA (or 0.5%formic acid) and then eluted with 10 μl of 60% acetonitrile. The elutedpeptide solution was dried and analyzed using MALDI mass spectroscopy(Mann M, et al. Ann. Rev. Biochem. 70:437-473, 2001).

The mass spectrogram of the 53 kDa spot (following purification andprotease digestion) consisted of over 20 tryptic peptides, of which 16peptides were from the 53 kDa protein while the remaining peptides weretrypsin autodigestion products (data not shown). The 53 kDa peptideswere further analyzed using a sequence database search sharewaresoftware program called ProFound™(http://www.proteomics.com/prowl-cgi/Profound.exe). PROFOUND was used tosearch public databases for protein sequences (e.g., non-redundantcollection of sequences at the US National Center for BiotechnologyInformation (NCBI)). The NCBI database contains translated proteinsequences from the entire collection of DNA sequences kept at GenBank,and also the protein sequences in the PDB, SWISS-PROT and PIR databases.

Using the ProFound™ program, the 53 kDa protein was identified asvimentin (Chan et al., Biochemistry 28: 1033-1039, 1989; Hale andMansfield, Nucleic Acids Res. 17(23): 10112, 1989) with a probabilityZ-score of 2.43, which is in the 99.9^(th) percentile, based on theanalysis of the 16 tryptic peptide sequences that covered 43% ofvimentin's complete amino acid sequence (FIG. 12). Two values were takeninto account when evaluating the PROFOUND analysis result: the Zprobability score and the percent coverage (the percent of the peptides'amino acid sequences relative to the identified protein's complete aminoacid sequence). Z=1.65-2.43 is an acceptable range of scores. Z=1.65means that the result is in the 95th percentile and Z=2.43 means thatthe result is in the 99.9th percentile. Thus the Z score of 2.43indicated that the 53 kDa spot peptide sequences corresponded to thoseof vimentin with the highest degree of probability.

The sequences of the 16 peptides were spread throughout the vimentinmolecule and together corresponded to 43% of the complete vimentinprotein amino acid sequence, hence the protein identified was the fulllength protein and not a fragment or fusion protein.

To confirm that vimentin is overexpressed in MCF-7/AR cells totalextracts were resolved by 2D-PAGE, transferred onto nitrocellulose andblotted with anti-vimentin (MS-129-P, NeoMarker).

As shown in FIGS. 2(A) and (B), Vimentin was overexpressed in the totalcell extracts of multidrug resistant MCF-7/AR cells by at least a factorof 11 as compared to the total cell extracts of drug-sensitive MCF-7cells.

Example 3 5.3 Cell Surface Expression of Vimentin in MDR LymphoblasticLeukemia Cancer Cells

To determine whether vimentin was present on the inside or outside ofthe cell membrane, intact lymphoblastic leukemia tumor cells (CEM andCEM/VLB) were treated the same way as in Example 2, with a membraneimpermeable biotinylating reagent that reacts with lysines, and totalcell extracts from both drug sensitive (CEM) and multidrug resistant(CEM/VLB) were prepared. Two equivalent sets of 2-D gels of CEM andCEM/VLB were prepared and silver stained or transferred ontonitrocellulose and blotted with streptavidin-HRP.

FIGS. 3A and B represent the 2-D streptavidin-HRP blot of CEM andCEM/VLB total surface biotinylated cell extracts, respectively the arearepresented corresponds to the presumed location of vimentin on a 2-Dgel (MW=53 kDa, pI=5.1). The 50 kDa protein was overexpressed on thesurface of multidrug resistant CEM/VLB cells by at least a factor of twoas compared to the drug-sensitive CEM cells (compare FIGS. 3A and B).

To confirm the identity of this protein as vimentin, the 50 kDa spot waslocalized on the silver stained gel using the coordinates obtained fromthe corresponding blot, was excized and processed to prepare a samplefor MALDI analysis as described in example II. The mass spectrogram ofthe 50 kDa spot (following tryptic digestion and peptide purification)consisted of over 20 tryptic peptides, of which 15 peptides were fromthe 50 kDa protein while the remaining peptides were trypsinautodigestion products. The 50 kDa peptides were further analyzed usingthe sequence database search shareware software program called ProFound™(http://www.proteomics.com/prowl-cgi/Profound.exe).

The 50 kDa protein was identified as vimentin (Chan et al., Biochem. 28:1033-1039, 1989; Hale and Mansfield, Nucleic Acids Res. 17(23): 10112,1989) with a probability Z-score of 2.43, which is in the 99.9^(th)percentile, based on the analysis of 14 tryptic peptide sequences thatcovered 52% of its complete amino acid sequence. The Z score of 2.43indicated that the about 50 kDa spot peptide sequence corresponded tothose of vimentin with the highest degree of probability.

Full length vimentin protein was expressed on the cell surface ofCEM/VLB cells and was biotinylated at one site: at a lysine residuelocated relatively close to the C-terminus of the molecule. Thesequences of the 15 peptides were spread throughout the vimentinmolecule and together corresponded to 52% of the complete vimentinprotein amino acid sequence, hence the protein identified was, again,the full length protein and not a fragment or fusion protein. Thebiotinylated region was detected in two ways. First, the bound biotinmoiety changed the mass of the peptide containing it relative to theunbiotinylated peptide, and secondly, biotinylated peptides bound tostreptavidin beads (immobilized streptavidin beads commerciallyavailable from Pierce #20347, Dallas, Tex.), whereas non-biotinylatedpeptides did not.

Example 4 5.4 Cell Surface Expression of Vimentin in MDR PromyelocyticLeukemia Cancer Cells

To determine whether vimentin was present on the inside or outside ofthe cell membrane, intact promyelocytic leukemia tumor cells (HL60 andHL60/AR) were reacted with a membrane impermeable biotinylating reagentthat reacts with the amino acid lysine, and total cell extracts fromboth drug sensitive (HL60) and multidrug resistant (HL60/AR) wereprepared.

To do this, cells were biotinylated by washing 3× with 50 ml PBS, pH 8.Next, Sulfo-NHS-LC-LC-Biotin (Pierce Chemicals, Dallas, Tex.), which isa membrane impermeable reagent, was prepared at 0.1-0.5 mg/ml and addedto cells. The incubation with Sulfo-NHS-LC-LC-Biotin was allowed tocontinue for 1 hour at 4° C. with rotation. The reaction was stopped bywashing cells one time with 50 ml PBS pH 8, containing 10 mM glycine andseveral times with 50 ml PBS, without glycine. Cells were then lysed in200 μl of buffer A (1% SDS and 0.05 M Tris/HCl, pH 7.4), containingproteases inhibitors (Proteases inhibitors: 1 μg/ml pepstatin, 1 μg/mlLeupeptin; 1 μg/ml benzamidine; 0.2 mM PMSF) and incubated 5 minutes onice. The cell lysate was then sonicated with a Vibracell sonicatoramplitude 40 setting #25 for 3×10 seconds with 1 minute on ice betweenshots. The sonicated cell lysate was mixed with 800 μl of buffer B(1.25% Triton-X100, 0.05 M Tris/HCl, pH 7.4, 190 mM NaCl), containingproteases inhibitors and incubated 5 minutes on ice. The cell lysate wasnext centrifuged at 14,000 rpm in an Eppendorf microfuge for 5 minutes.The supernatant was removed, and its protein concentration wasdetermined using the DC protein assay kit from BIORAD according tomanufacturer's instructions (BioRad Laboratories, Hercules, Calif.) (seealso Lowry et al., J. Biol. Chem. 193: 265-275, 1951).

In addition, streptavidin purified biotinylated proteins were preparedfrom the surface-biotinylated total cell extracts of HL60 and HL60/ARcells using immobilized streptavidin (commercially available fromPierce, catalog no #20347 or Amersham Pharmacia Biotech RPN1231(Piscataway, N.J.).) To do this, 50 μl samples containing 500 μg to 2 mgprotein were diluted to 450 μl final with buffer C (1:4 v/v of buffers Aand B above), containing proteases inhibitors. Samples were thencentrifuged at 14,000 rpm in an eppendorf microfuge for 1 minute. Thesupernatant was transferred to a new Eppendorf tube and mixed with 100μl of Streptavidin-linked sepharose beads. The protein lysate togetherwith the linked sepharose beads were incubated with rotation overnightat 4° C. The mix was centrifuged at 14,000 rpm for 30 seconds in aneppendorf microfuge. The supernatant was removed and the protein loadedbeads were washed 3 times with buffer C, then with 500 mM NaCl in bufferC and buffer C again. Proteins were eluted from the Streptavidin-linkedbeads with SDS sample buffer following 10 minutes boiling. Elution wasrepeated and volumes pooled.

Equivalent amounts of protein from HL60 and HL60/AR (promyelocyticleukemia) cell surface biotinylated total cell extracts and streptavidinpurified cell surface biotinylated extracts were resolved by SDS-PAGEaccording to the method of Laemmli (supra) and subjected to Westernblotting analysis and probed with either anti-vimentin antibody (mousemonoclonal MS-129-P, NeoMarker) or with horseradish peroxidase(HRP)-linked streptavidin (which specifically binds to biotinylatedproteins, Amersham RPN 1231). To do this, gels containing separatedproteins were transferred onto Hybond C nitrocellulose membrane(Amersham, Piscataway, N.J.) according to the method of Towbin (Towbin,H. T., Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979). Thenitrocellulose membranes were then probed with antibody orHPR-streptavidin. Binding of the antibody was detected with HRPconjugated goat anti-mouse secondary antibody (BioRad). Both secondaryantibody and HRP-linked streptavidin were detected using the ECLchemiluminescent detection kit commercially available from Pierce.Relative protein levels were detected by exposure in the dark to XARfilms (Kodak, Rochester, N.Y.).

Anti-vimentin antibody bound to vimentin protein that was expressed atsignificantly higher levels in surface biotinylated total cell extractsof multidrug resistant HL60/AR cells compared to HL60 cells (see FIG.4A). Vimentin was also overexpressed in streptavidin purified cellsurface biotinylated proteins of HL60/AR cells compared to HL60 cells(see FIG. 4B).

To confirm that vimentin present on the cell surface was indeedbiotinylated, surface biotinylated total cell extracts from HL60 andHL/60AR cells were immunoprecipated with anti-vimentin antibody and theimmunoprecipates were resolved on SDS-PAGE and Western blotted. To dothis, samples were prepared as described above using Protein A Sepharosebeads instead of streptavidin beads. To elute the proteins from theProtein A Sepharose beads, the loaded beads were washed five times withBuffer D (0.03% SDS, 0.05 M Tris-HCl, pH 7.4, 0.1% Triton X-100, 5 mg/mlBSA fraction V, 150 mM NaCl) and one time with Buffer E (150 mM NaCl,0.05 M Tris-HCl, pH 7.4), containing protease inhibitors as above.Proteins were eluted from the beads with SDS sample buffer following 10minutes incubation at room temperature with vortex every 1 minute.Protein elution from the beads was repeated one more time and thevolumes pooled. Proteins were resolved by SDS-PAGE and Western blottingas before with either anti-VIM monoclonal antibody or HRP-labeledstreptavidin.

The blots were probed with anti-vimentin antibody and withstreptavidin-HRP. As expected, vimentin was detected in theimmunoprecipitates from both cell types, however, significantly morecell surface biotinylated vimentin was present in the anti-vimentinimmunoprecipitates from HL60/AR cells compared with those from HL60cells (see FIGS. 4C and 4D). Vimentin has a highly sensitive proteasereactive site at D90 which may account for a lower molecular weight bandobserved when vimentin was immunoprecipitated with anti-vimentin.

These results, taken together, suggest that translocation of vimentinacross the plasma membrane to the cell surface, as well as additional denovo synthesis of vimentin, was associated with multidrug resistance inHL60/AR cells.

Example 5 5.5 Cell Surface Expression of Vimentin in MDR Breast Cancer

To determine whether vimentin was present on the inside or outside ofthe cell membrane, intact breast tumor cells (MCF-7 and MCF-7/AR) weretreated with a membrane impermeable biotinylating reagent that reactswith the amino acid lysine, and total cell extracts from both drugsensitive (MCF-7) and multidrug resistant (MCF-7/AR) were prepared. Inaddition, streptavidin purified biotinylated proteins as well asanti-vimentin immunoprecipates were prepared from the surfacebiotinylated total cell extracts of MCF-7 and MCF-7/AR cells, resolvedon SDS-PAGE and transferred onto nitrocellulose the same way as inExample 4.

Anti-vimentin antibody bound to vimentin protein which was expressed atsignificantly higher levels in surface biotinylated total cell extractsof multidrug resistant MCF-7/AR cells compared to MCF-7 cells (see FIG.5A). Vimentin was also overexpressed in streptavidin purified cellsurface biotinylated proteins of MCF-7/AR cells compared to MCF-7 cells(see FIG. 5B). In addition, the immunoprecipitate blots were probed withanti-vimentin antibody and with streptavidin-HRP. As expected,significantly more cell surface biotinylated vimentin was present in theanti-vimentin immunoprecipitates from MCF-7/AR cells compared with thosefrom MCF-7 cells (see FIGS. 5C and 5D).

These results, taken together, suggest that translocation of vimentinacross the plasma membrane to the cell surface, as well as additional denovo synthesis of vimentin, was associated with multidrug resistance inMCF-7/AR cells.

Example 6 5.6 Cell Surface Expression of Vimentin in MDR PromyelocyticLeukemia Cancer Cells

HL60 and HL60/AR cells were analyzed by cell surface staining andfluorescence-activated cell sorter (FACS) analysis to determine thedifference in cell surface expression of vimentin on the two cell lines.To do this, indirect immunofluorescence analysis was performed using 1μg of rabbit polyclonal anti-vimentin as primary antibody (CBL46, CymbusBiotech, Hampshire, UK), followed by goat anti-rabbit IgGFITC-conjugated secondary antibody (Cat # F9887, Sigma).

The following indirect staining procedure was used to prepare the cellsfor FACS analysis:

Cells were washed three times in 50 ml PBS, pH 7.4 and 0.1% NaN₃ andcounted.

1×10⁶ cells per sample were placed in 100 μl PBS and 0.1% NaN₃ in 12×75mm tubes or deep 96 well plate. The first antibody (rabbit polyclonalanti-vimentin antibody) was added and incubated for 20 minutes at 37° C.3 ml (or 1.25 ml in plate) PBS pH 7.4 and 0.1% NaN₃ was added themixture was spun for 5 min (1000 rpm/200×g). The supernatant wasdiscarded and the pellet was resuspended in 100 μl PBS pH 7.4 and 0.1%NaN₃.

The second Ab (FITC-conjugated) was added. The mixture was prepared bydiluting ½ in PBS, pH 7.4 and 0.1% NaN₃ and spun at maximum speed for 30min. at 4° C. Separate from pellet and use a 1/10 for staining. Incubatefor 20 min at 37° C. 3 ml (or 1.25 ml in plate) PBS pH 7.4 and 0.1% NaN₃was added and the mixture spun 5 min (1000 rpm/200×g). The supernatantwas discarded and the addition of PBS and NaN₃ was repeated. The mixturewas spun again for 5 min (1000 rpm/200×g).

1 μg-2 μg/μl of EMA was added. The mixture was incubated in white lighton ice for 10 minutes. 3 ml (or 1.25 ml in plate) PBS pH 7.4 and 0.1%NaN₃ was added and the mixture spun 5 min (1000 rpm/200×g). Thesupernatant was discarded and the addition of PBS and NaN₃ was repeated.The mixture was spun again for 5 min (1000 rpm/200×g). Resuspend in 500μl PBS pH 7.2/2% paraformaldehyde and store in the dark at 4° C.

For each sample, 10,000 cells were analyzed using afluorescence-activated cell sorter (Beckman Coulter XL MCL). Thefluorescence emission corresponding to specifically stained cells wascalculated by subtracting the emission measured for cells at 530 nmstained with precleared rabbit IgG negative control. In some cases,rabbit IgG controls were precleared with HL60 or HL60/AR cells in orderto reduce the non-specific staining on each cell line. This was done byincubating 1 μg of rabbit IgG with 1-2×10⁶ cells in 50 μl-100 μl PBS for20 min at 37° C. The mix was then spun and the supernatant used asnegative control or carried through one or two additional incubations.The supernatant was used as rabbit IgG control for HL60 and HL60/AR whenincubated with HL60 and HL60/AR respectively.

The results showed that vimentin was overexpressed on the surface ofHL60/AR cells at a level five fold higher than the level of vimentinexpressed on the surface of HL60 cells (see FIG. 6).

As expected, the difference between the expression level of vimentin onthe surface of HL60/AR cells and the expression level of vimentin on thesurface of HL60 cells was better observed when precleared rabbit IgGcontrols were used. Additionally, the difference between the expressionlevel of vimentin on the surface of HL60/AR cells and the expressionlevel of vimentin on the surface of HL60 cells was also better observedwhen low levels of primary antibody were used. Thus, when indirectimmunofluorescence analysis was performed using 0.1 μg rather than 1 μgof polyclonal anti-vimentin as primary antibody (CBL46, Cymbus Biotech,Hampshire, UK) and 0.1 μg rather than 1 μg of precleared ornon-precleared rabbit IgG as negative control, the fold difference inexpression levels of vimentin on HL60/AR versus HL60 cells was 26(precleared) and 9 (non-precleared) respectively.

In addition, indirect immunofluorescence analysis was performed usingincreasing amounts 10 μg-20 μg-40 μg of monoclonal anti-VIM as primaryantibody (commercially available from NeoMarker, catalog no. MS-129-P),followed by anti-mouse IgG-FITC conjugated secondary antibody at 1:10dilution (commercially available from Chemicon, catalog #AP181F) Thefluorescence emission corresponding to specifically stained cells wascalculated by subtracting the emission measured for cells at 530 nmstained with mouse IgG₁ isotype control. 40 μg of anti-VIM wassaturating for HL60/AR.

Similarly, experiments were performed with amounts of antibody rangingfrom 2.5 μg to 40 μg to determine the concentration of anti-vimentinthat would be saturating HL60 cells. The values of fluorescenceintensities obtained didn't increase in a concentration dependent mannerand ranged between 0.2 and 0.9 RFI indicating that the cells werealready saturated at 2.5 μg. 40 μg of anti-vimentin were therefore usedto quantify the number of molecules of vimentin present at the surfaceof HL60 and HL60/AR cells.

At saturating concentration (40 μg of monoclonal anti-vimentin),vimentin was expressed on the surface of HL60/AR cells at a level37-fold higher than the level of vimentin expressed on the surface ofHL60 cells. The number of molecules of vimentin expressed on the surfaceof HL60/HL60/AR cells has been determined using a quantum simplycellular flow cytometry quantification kit (Serotec #FCSC814: 498-44298molecules, Raleigh, N.C.). The QSC system consists of microbeads ofapproximately the size of lymphocytes that are coupled to goatanti-mouse antibodies. A set of five populations of microbeads bearingdifferent known amounts of goat anti-mouse antibodies are provided inthe kit. The microbeads are incubated with saturating amounts of themouse monoclonal antibody of interest. The fluorescence intensitiesobtained with the microbeads are plotted to create a standard curve ofthe fluorescence intensity to the number of antibody molecules bound onthe beads. The signal obtained with the cells (at saturating amounts) isthen, using the standard curve, correlated to the number of antibodymolecules bound to the cells, which corresponds to the number ofantigens present on the surface of the cell. At saturating conditions ofantibody for the beads and the cell lines, HL60 and HL60/AR have beenshown to bear approximately 186 and 5052 molecules of vimentinrespectively when using the mouse monoclonal anti-vimentin purchasedfrom NeoMarker (see FIG. 7). This corresponds to a greater than 27 foldincrease in cell surface vimentin expression in the multidrug resistantHL60/AR cell line as compared to the corresponding non-MDR HL60 cellline.

Example 7 5.7 Characterization of Cell Surface Vimentin Expression inMDR Lymphoblastic Leukemia Cancer Cells

Molt4 and Molt4/DOX cells were analyzed by cell surface staining andfluorescence-activated cell sorter (FACS) analysis to determine thedifference in cell surface expression of vimentin on the two cell lines.To do this, indirect immunofluorescence analysis was performed using 20μg of mouse monoclonal anti-vimentin as primary antibody (MS-129-P,NeoMarker), followed by goat anti-mouse IgG FITC-conjugated secondaryantibody (Chemicon, catalog #AP181F).

The cells were prepared according to the procedure set forth in Example6 above. For each sample, 10,000 cells were analyzed using afluorescence-activated cell sorter (Beckman Coulter XL MCL, Fullerton,Calif.). The fluorescence emission corresponding to specifically stainedcells was calculated by subtracting the emission measured for cells at530 nm stained with 20 μg of mouse IgG₁ isotype control.

The results of the FACS analysis demonstrate that vimentin was expressedon the surface of Molt4/AR cells at a level 1.5 fold higher than thelevel of vimentin expressed on the surface of Molt4 cells (see FIG. 8).

Example 8 5.8 Characterization of Cell Surface Vimentin Expression inMDR Breast Cancer Cells

HS574M, MCF-7, MCF-7/AR, MDA, MDA/AR and MDA/MITO breast cancer cellswere analyzed by cell surface staining and fluorescence-activated cellsorter (FACS) analysis to determine the difference in cell surfaceexpression of vimentin on the two cell lines. To do this, indirectimmunofluorescence analysis was performed using 5 and/or 20 μg of mousemonoclonal anti-vimentin as primary antibody (MS-129-P, NeoMarker),followed by goat anti-mouse IgG FITC-conjugated secondary antibody(Chemicon, catalog #AP181F, Temecula, Calif.).

The cells were prepared according to the procedure set forth in ExampleVI above. For each sample, 10,000 cells were analyzed using afluorescence-activated cell sorter (Beckman Coulter XL MCL). Thefluorescence emission corresponding to specifically stained cells wascalculated by subtracting the emission measured for cells at 530 nmstained with 5 μg or 20 μg of mouse IgG₁ isotype control.

The results of FACS analysis demonstrate that while vimentin was notexpressed on the surface of HS574M cells, it was expressed on thesurface of MCF-7/AR cells at a level 1.5 fold higher than the level ofvimentin expressed on the surface of MCF-7 cells (see FIG. 9).Similarly, vimentin was expressed on the surface of MDA/AR cells at alevel 3 to 5 fold higher than the level of vimentin expressed on thesurface of MDA cells (see FIG. 9). Vimentin was expressed on the surfaceof MDA/MITO cells at a level 3 folds higher than the level of vimentinexpressed on the surface of MDA cells (see FIG. 10).

The number of molecules of vimentin expressed on the surface of MCF-7and MCF-7/AR cells was determined using a quantum simply cellular flowcytometry quantification kit (Serotec #FCSC814: 498-44298 molecules) thesame way as described in example 5.6. At saturating conditions ofantibody for the beads and the cell lines (50 μg), MCF-7 and MCF-7/ARwas shown to bear approximately 14,556 and 22,010 molecules of vimentinrespectively when using the mouse monoclonal anti-vimentin purchasedfrom NeoMarker, which corresponds to a 1.5 fold difference (see FIG.11).

Example 9 5.9 Comparison of Vimentin Expression in Normal, Neoplasticand MDR Neoplastic Cell

Normal cells were next compared to hematological cancer cells and MDRhematological cancer cells to determine the difference in levels of cellsurface expressed vimentin. To do this, human blood samples (collectedin heparin tubes) were obtained from donors and were processed within anhour. Briefly, erythrocytes were separated from leukocytes and plasma ona Ficoll hypaque gradient (Histopaque Sigma 1077-1, St. Louis, Mo.).

Specifically, 15 ml of blood was diluted one-half in phosphate bufferedsaline (PBS), pH 7.4 and put over equal volume of Ficoll gradient. Cellswere separated by centrifugation at 400×g for 30 min. at roomtemperature. The upper phase (plasma) was removed until 0.5 cm from theplasma/Ficoll interface. Then, mononuclear cells (at the interface) werecollected in a 50 ml Falcon tubes. The Ficoll was removed and red bloodcells (at the bottom of the tube) were collected. All cells types werewashed with PBS two times by spinning for 10 min. at 250×g at 4° C. andcounted. Cells were then re-suspended at 1×10⁷ cells/ml in PBS, and 100μl (1×10⁶ cells) aliquots were used for flow cytometry (FACS) analysis.

The cells were stained with primary and secondary antibody for FACSanalysis as described in Example 6 above. For each sample, 10,000 cellswere analyzed using a fluorescence-activated cell sorter (BeckmanCoulter XL MCL). The fluorescence emission corresponding to specificallystained cells was calculated by subtracting the emission measured forcells at 530 nm stained with an isotype matching control.

Each FACS experiment was carried out with several controls, includingcells alone to determine autofluorescence; cells plus EMA to identifydead cells during analysis; cells plus secondary antibody (Ab) alone toidentify non-specific interactions due to secondary antibody; cells plusisotype matching antibodies (Abs) or appropriate host primary Abscontrol; cells (mononuclear cells) plus CD45-PC5 mouse monoclonal,phycoerythrin-Cyanine 5 conjugate (Immunotech PN IM2653) to establishand gate the different leukocyte populations; and erythrocytes plusGlycophorin A-FITC mouse monoclonal, Cymbus CBL 409F as positive controlfor erythrocytes.

When red blood cells or white blood cells from normal, healthy donorswere stained with 0.25 μg-0.5 μg or 1 μg (3, 6 and 21 donors,respectively) of polyclonal anti-vimentin antibody (CBL46, CymbusBiotech), in contrast to HL60 and HL60/AR stained with 1 μg ofpolyclonal anti-vimentin antibody, the normal RBC and WBC were found notto express any vimentin on their cell surface (results not shown).

Therefore, as well as showing an increase in MDR neoplastic versusnon-MDR neoplastic cells of the same type, cell surface vimentinexpression is increased in neoplastic versus non-neoplastic cells of thesame or similar origin. Accordingly, levels of cell surface vimentin maybe used to detect and or diagnose neoplasms as wells as MDR neoplasms.

Example 10 5.10 Vimentin-Based Vaccine Protection Against MDRHematological Cancer Cells and MDR Mammary Adenocarcinoma Cells

To determine whether or not the full length vimentin protein expressedon the cell surface of MDR hematological cancer cells is useful as anantigen for a vaccine to immune animals against MDR hematological cancercells, purified vimentin protein is combined with an adjuvant (e.g.,Freund's), and administered to groups of mice having a hematologicalcancer caused by the presence of a hematological cancer cell. One suchnon-limiting hematological cancer is Acute lymphocytic leukemia.

To do this, the mice are injected with hematological tumor cells thatare compatible with the mice's MHC type (or are injected into SCIDmice). Some of the mice receive the injected tumor cells prior to beingimmunized with purified full length murine vimentin protein; somereceive the hematological tumor cell injection after being immunizedwith purified full length murine vimentin protein. Note that thepurified vimentin protein may be administered with an adjuvant. Propercontrols are performed for each group of mice (i.e., one control groupreceives only the purified murine vimentin protein; another receivesonly the hematological tumor cell injection).

The tumors that form in the mice are weighed or measured (e.g., tumorcell number counted, tumor excised and weighed, or tumor measured bycalipers). The mice that are vaccinated with vimentin prior to injectionof the tumor cells are found to have tumors that are smaller aftertreatment than those that were not vaccinated with vimentin prior toinjection of the tumor cells.

In further studies, the efficacy of vimentin as an antigen against MDRmammary adenocarcinoma cells (MCF/AR) is assessed. Briefly, six week-oldfemale mice are injected with 30-250 ug of whole vimentin or controlantigen administered with or without adjuvant S.C. and I.P or into rearfootpads on days 1, 7, 14, 21, 28 and 35. After various intervals, bloodsamples are collected from the retro-orbital venous plexus foranti-vimentin antibody assay. Mice are challenged on day 59 with 1.5×10⁴viable MDR mammary adenocarcinoma cells (MCF/AR) administered S.C. intothe right flauk. Mice are examined twice a week and tumor incidence isdetermined from the number of mice bearing tumors. Tumor size wasmeasured with a Vernier caliper. Survival rates are measured up to 80days post challenge with adenocarcinoma.

Example 11 5.11 Vimentin-Targeted Therapy Against MDR HematologicalCancer Cells

In order to determine whether targeting a therapeutic to cell surfacevimentin would be useful in treating a preexisting cancerous condition,hematological tumor cells are administered to MHC-matched mice, andtumors are allowed to form. Next, the mice are administered vincristine(or another chemotherapeutic drug) at a dosage predicted to kill most,but not all of the tumor cells in the mice. Those mice that areidentified as having developed multidrug resistant tumor cells areadministered a composition comprising vincristine and a binding agentthat specifically binds to murine vimentin protein, where the bindingagent is operably linked to ricin toxin.

The mice that receive the composition show a better prognosis (i.e.,smaller tumor or fewer tumor cells) as compared to mice that receiveonly the binding agent or only the vincristine.

In further studies, the efficacy of a vimentin-targeted therapeutic intreating an MDR mammary adenocarcinoma cells (MCF/AR) is assessed.Briefly, Athymic nude mice are used for the MCF-7/ADR xenografts. Malemice 5-7 weeks old, weighing 18-22 g, are used. Mice receive asubcutaneous (s.c.) injection of the cells using 0.5 millioncells/inoculation under the shoulder. After s.c. implantation of thecells, when the s.c. tumour is approximately 5.5 mm in size, mice arerandomized into treatment groups of four including controls and groupsreceiving vincristine or doxorubicin alone (4 mg/kg), intraperitoneally(i.p.) every 2 days, anti-vimentin alone (100 ug-1 mg/kg) or bothvincristine or doxorubicin and anti-vimentin mAb (100 ug-1 mg/Kg), i.p.The animal's weight is measured every 4 days. Each animal is tagged inthe ear and followed individually throughout the experiments. Tumorgrowth starting on the first day of treatment is measured and the volumeof the xenograft is monitored every 4 days. The mice are anaesthetizedand killed when the mean tumor weights is over 1 g in the control group.Tumor tissue is excised from the mice and its weight is measured.

Example 12 5.12 Qualitative Analysis of Cell Surface Vimentin Expressionin MDR Breast Cancer Cells

MCF-7 and MCF-7/AR cells were analyzed by immunostaining to determinethe difference in cell surface expression of vimentin on the two celllines. To do this, immunofluorescence analysis was performed using 0.5μg of anti-vimentin as primary antibody (clone V9, NeoMarker MS-129-P)on permeabilized and non-permeabilized cells. Ethidium monoazide (EMA)was used to stain the nucleus of permeabilized or damaged cells.

FIGS. 13A and 13B provides flow charts depicting the steps taken tostain cells. The control cells were MCF-7 and MCF-7/AR stained with 0.5μg mouse IgG1 and no staining was observed in either the permeabilizedor non-permabilized cells (data not shown). As shown in FIG. 14,vimentin was clearly expressed on the surface of intact multidrugresistant MCF-7/AR cells while MCF-7 didn't show any staining. Whencells were permabilized, MCF-7 showed minimal staining whereas MCF-7/ARshowed a very abundant network of vimentin filaments (FIG. 14).

Example 13 5.13 Quantitative Analysis of Cell Surface VimentinExpression in MDR Promyelocytic Leukemia Cancer Cells

HL60 and HL60/AR cells were analyzed for surface exposure of vimentin bydirect binding of 125-iodine labeled anti-vimentin to the surface of thecells. To do this, anti-vimentin (NeoMarker, MS-129-PABX), mouse IgG1(isotype matching negative control, Sigma, M-9035) and anti-CD33(positive control, Serotec, #MCA1271) were iodinated using IODO-GEN®precoated iodination tubes (Pierce, #28601) following the proceduregiven by the manufacturer (average activity obtained: 7.5 μCi/μg).

Cells were washed twice with RPMI 1640 and resuspended at 10⁶ cells/100μL in the same media. Viability was assessed by trypan blue staining andwas less than 5%. Cells (1×10⁶) were aliquoted into borosilicate tubesand incubated for 1 hour on ice in the presence of 1 μg of radiolabeledanti-vimentin, IgG1 or anti-CD33. After incubation, cells were washed 3times with 1 ml RPMI 1640 and the cell pellet was counted in agamma-counter.

The results of the intact cell radioimmunoassay are represented in a bargraph in FIG. 15. The numbers are expressed in counts per minuteobtained for the cell pellet from which the IgG1 background has beensubtracted. Vimentin was expressed on the surface of the resistantHL60/AR cells at a level 3 folds higher than the level of vimentinexpressed on the surface of drug-sensitive HL60 cells.

Example 14 5.14 Cytotoxic Effect of Radioiodinated Anti-Vimentin on HL60and HL60/AR Cells

Cells were washed twice with RPMI 1640 and resuspended at 10⁶ cells/100μl in the same media. Viability was assessed by trypan blue staining andwas less than 5%. Cells (1×10⁶) were aliquoted into borosilicate tubesand incubated for 4 hour at 37° C. (0.5% CO₂) in the presence of 5 μg ofsterile filtered radiolabeled anti-vimentin, IgG1 or anti-CD33(iodination procedure was described in example 5.13). After incubation,cells were washed once with 1 ml RPMI 1640, 10% FBS, 1 mM HEPES,resuspended in 1 ml of the same media and seeded at 5000 cells per wellinto a flat bottom tissue culture 96 well plate. The cytotoxicity of theradiolabeled antibodies was assessed after 72 hours in an MTT basedassay. Anti-CD33 was used a positive control for surface binding of theantibody and 120 nM doxorubicin (Doxo) was used as positive control forcytotoxicity.

The results of the cytotoxicity assay are represented in a bar graph inFIG. 16. Values are expressed as percent viability. Values for percentsurvival with anti-vimentin and anti-CD33 antibody-¹²⁵I conjugates werenormalized against the values obtained for the radiolabeled IgG1(non-significant background binding, 100% viability). Values obtainedwith the anticancer drug doxorubicin were normalized against the valuesobtained for cells non-treated with drug.

The results obtained demonstrate that radiolabeled anti-vimentin has acytotoxic effect on HL60 and HL60/AR which both express vimentin ontheir surface. The slight increase in cytotoxicity observed for HL60/AR(10%) might be due to the fact that HL60/AR drug-resistant cells expressmore vimentin on their surface than HL60 drug-sensitive cells.

Example 15 5.15 Molecular Quantitation of Cell Surface Vimentin in DrugSensitive and Drug Resistant Breast and Ovarian Cancer Cell Lines

Breast MCF-7, MCF-7/AR, MDA, MDA/mito, and ovarian SKOV3, SKOV/T320(resistant to taxol) 2008 and 2008/T320 (resistant to taxol) cells wereanalyzed for surface exposure of vimentin by direct binding of125-iodine labeled anti-vimentin to the surface of the cells. To dothis, anti-vimentin (NeoMarker, #MS-129-PABX) and mouse IgG1 (isotypematching negative control Sigma, M-9035) were iodinated using IODO-GEN®precoated iodination tubes (Pierce, #28601) following the proceduregiven by the manufacturer (average activity obtained: 7.5 μCi/μg).

Cells were seeded at 20,000 cells per well into a 96 Stripwell™ plate(Costar, #9102). After an overnight growth in complete media (37° C.,0.5% CO₂), wells were gently washed with 100 μl media containing 1% FBSand incubated for 1 hour at 37° C. (0.5% CO₂) in 100 μL media containing1% FBS and 0.1% sodium azide as well as 100 ng of radioiodinatedanti-vimentin (or IgG1). Mortality was checked prior to incubation bytrypan blue staining and was typically less than 1%. After incubation,media was discarded, wells washed twice with 350 μL media containing 1%FBS, and individually counted in a gamma-counter.

Binding studies were performed on MDA/mito cells using increasingamounts of radiolabeled anti-vimentin in an intact cellradioimmunoassay. Scatchard analysis was used to calculate the apparentdissociation constant (K_(d)) and the number of molecules of antibodybound per cell. The average number of molecules of anti-vimentin boundper cell was obtained from the average of 5 independent Scatcharddeterminations and is reported in the table in FIG. 17A. FIG. 17B showsa Scatchard plot for the first experiment described in the table in FIG.17A.

The average number of vimentin epitopes present on the surface ofMDA/mito cells was 2.49×10⁶ per cell and the average K_(d) was 5.7 nM.The K_(d) was determined for MDA cells in a separate experiment and was9.3 nM (see Table in FIG. 17C), which is equivalent to the K_(d)measured for MDA/mito cells.

The number of vimentin epitopes present on MCF-7, MCF-7/AR, MDA, SKOV3,SKOV/T320, 2008 and 2008/T320 was calculated from the fold difference insurface exposure between these cell lines and MDA/mito. This fold wasdetermined by testing the same number of MDA/mito cells and cells listedabove (about 20,000 cells) using the above intact cell radioimmunoassaywith 100 ng of radiolabeled anti-vimentin. The latter numbers of surfacevimentin epitopes represent the average from multiple independentexperiments (n=2 to 5). These numbers are reported in the table in FIG.17C and show that MCF-7/AR, MDA, MDA/mito, SKOV3 and SKOV/T320 cellsexpress large amounts of vimentin on their surface (up to 2.5×10⁶ forMDA/mito), whereas MCF-7, 2008 and 2008/T320 numbers are relatively low(note that “2.5.E+06 represents base 10 Exponential, i.e. 2.5×10⁶). Thefolds difference in surface exposure of vimentin between sensitive andresistant counterpart cells are given as R/S and were 41.2, 4, 1.6, and2 for MCF-7/AR, MDA/mito, SKOV/T320 and 2008/T320 respectively.

Example 16 5.16 Induction of Cell Surface Exposure of Vimentin in Breastand Ovarian Cancer Cells

Given the findings reported in example 5.15 above, which shows thatvarious breast and ovarian cancer cells selected with doxorubicin,mitoxanthrone or taxol demonstrate increased surface exposure ofvimentin when compared to their sensitive counterpart, it was ofinterest to determine whether these drugs were capable of inducingsurface exposure of vimentin in MDA and SKOV3 cell lines. To do this,20,000 cells were seeded into a 96 Stripwell™ plate (Costar, #9102).After 5 h, the culture media was removed and replaced with culture mediacontaining various drugs (as indicated in FIG. 18) and incubated for 12to 16 hours at 37° C. (0.5% CO₂). After incubation, wells were gentlywashed with 100 μL media containing 1% FBS and incubated for 1 hour at37° C. (0.5% CO₂) in 100 μL media containing 1% FBS and 0.1% sodiumazide as well as 100 ng of radioiodinated anti-vimentin (or IgG1)(labeling procedure is given in example 5.13). Mortality was checkedprior to incubation by trypan blue staining and was typically less than1%. After incubation, media was discarded, wells washed twice with 350μL media containing 1% FBS, and individually counted in a gamma-counter.FIG. 18 represents the surface exposure of vimentin when MDA cells wereincubated with 1 or 10 μM taxol, 1 or 10 μM doxorubicin, or 0.1 or 1 μMmitoxanthrone. Values were corrected for non-specific binding with IgG1.A slight induction of surface exposure of vimentin was observed with alldrugs, mitoxanthrone having the most significant effect. A similar butless intense effect was observed when SKOV3 cells were treated with 1 μMtaxol. These finding are of importance in the context of immunotherapycombined with drugs.

Example 17 5.17 Internalization of ¹²⁵I-labeled Anti-Vimentin byMCF-7/AR, MDA, MDA/AR and MDA/Mito Cells

Internalization of cell surface vimentin was measured on breast cancercells kept in suspension (see FIG. 19A) or cells adhered in a 96 wellplate (FIG. 19B). FIG. 19A, shows 10⁶ cells were subcultured with adissociation buffer (Gibco, #13150-016), and then transferred to aborosilicate tube, washed in PBS and resuspended in 200 μl alpha-MEM, 3%BSA containing 1 μg of radioiodinated anti-vimentin, or 1 μg ofanti-Mucin-1 as positive control for internalization or 1 μg of IgG1 asbackground control. Mucin-1 (CD227) is a highly glycosylated proteinubiquitously present in many human tissues that, in tumor cells, isoften produced at elevated levels and with an abnormal glycosylationpattern. Mouse antibodies have been used in clinical trials for thepurpose of treating such cancers and are commercially available (e.g.,from Fitzgerald Industries, Inc., Concord, Mass.). After 1 hourincubation at 4° C., cells were washed twice and further incubatedadditional 4 hours at 4° C. or 37° C. Cell viability was 100% accordingto trypan blue staining of cells prior to incubation with radiolabeledIgGs. After incubation, cells were washed and the radiolabel determinedby counting samples in a gamma-counter. Cells were then stripped for 10min at RT with 50 mM L-Gly, pH 2.8, 150 mM NaCl, washed and counted forresidual activity in a gamma counter. The graph shown in FIG. 19Arepresents the percent of surface associated radiolabel (stripped withL-Gly) and internalized (remaining after stripping) for MCF-7/AR cellsat 4° C. and 37° C. The % internalization was obtained from thedifference in internalization measured at 4 and 37° C. and was 38.2% forvimentin and 12.5% for Mucin-1.

The results show that anti-vimentin antibodies attached to aradionuclide therapeutic agent (or diagnostic probe) bind to cellsurface vimentin present on the surface of multidrug resistant breastepithelial neoplastic cells, and are actively internalized in atemperature-dependent manner. The temperature dependence suggests thatthe antibody-¹²⁵I conjugate is actively taken up by endocytosis.Regardless of the mechanism of uptake, the results indicate thatanti-vimentin antibodies are capable of recognizing and transportingtherapeutic agents (or diagnostic agents) into MDR neoplastic cells forthe treatment (or diagnostic detection) of such cells.

For FIG. 19B, cells were prepared the same way as for an intact cellradioimmunoassay (20,000 cells per well). Briefly, after an overnightgrowth, wells were washed and preincubated in 100 μl media+1% FBScontaining 100 ng of radioiodinated anti-vimentin or IgG1 as backgroundcontrol, for 1 hour at 4° C. Mortality was checked prior topreincubation by trypan blue staining and was less than 1%. After thepreincubation, media was discarded, wells washed twice with 350 μL mediacontaining 1% FBS, and further incubated for 4 hours at 4° C. or 37° C.After incubation, wells were washed the same way as earlier and cellswere stripped for 10 min at RT with 50 mM L-Gly, pH 2.8, 150 mM NaCl,and washed. Stripped material and wells were then counted for activityin a gamma counter. The graph in FIG. 19B represents the percent ofsurface associated cpm (stripped with L-Gly) and internalized (remainingafter stripping) for MCF-7/AR, MDA, MDA/AR, MDA/mito cells at 4 and 37°C. The percent internalization of vimentin was obtained from thedifference in internalization measured at 4 and 37° C. and was 11.3,25.3, 24.8 and 8.9% for MCF-7/AR, MDA, MDA/AR and MDA/mito respectively.

These results further support the usefulness of anti-vimentin antibodiesfor the targeting and uptake of linked therapeutic and diagnostic agentsfor both neoplastic, and MDR neoplastic cells.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

1.-9. (canceled)
 10. A method for detecting a multidrug resistant cellin a patient comprising: (a) administering to the patient, a vimentinbinding agent operably linked to a detectable label; and (b) detectingthe label operably linked to the vimentin binding agent, wherein thevimentin binding agent specifically binds to cell surface-expressedvimentin present on a multidrug resistant cell in the patient.
 11. Themethod of claim 10, wherein the vimentin binding agent is an antibody orfragment thereof.
 12. The method of claim 10, wherein the vimentinbinding agent is selected from the group consisting of modified LDL,NLK1 protein, vimentin, desmin, glial fibrillary acidic protein, andperipherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase2A.
 13. The method of claim 10, wherein the vimentin binding agent isselected from the group consisting of natural ligands, synthetic smallmolecules, chemicals, nucleic acids, peptides, proteins, and antibodies.14. The method of claim 10, wherein the detectable label is selectedfrom the group consisting of fluorophores, chemical dyes, radioactivecompounds, chemoluminescent compounds, magnetic compounds, paramagneticcompounds, promagnetic compounds, enzymes that yield a colored product,enzymes that yield a chemoluminescent product, and enzymes that yield amagnetic product.
 15. The method of claim 14, wherein the multidrugresistant cell is a neoplastic cell.
 16. The method of claim 15, whereinthe neoplastic cell is selected from the group consisting of a breastcancer cell, an ovarian cancer cell, a myeloma cancer cell, a lymphomacancer cell, a melanoma cancer cell, a sarcoma cancer cell, a leukemiacancer cell, a retinoblastoma cancer cell, a hepatoma cancer cell, aglioma cancer cell, a mesothelioma cancer cell, and a carcinoma cancercell.
 17. The method of claim 15, wherein the neoplastic cell isselected from the group consisting of a promyleocytic leukemia cell, a Tlymphoblastoid cell, a breast epithelial cell, and an ovarian cell. 18.The method of claim 10, wherein the patient is a human.
 19. The methodof claim 18, wherein the patient is suffering from a disease or disordercaused by the presence of the multidrug resistant cell.
 20. A kit fordiagnosing or detecting multidrug resistance in a test neoplastic cellcomprising: a) a first probe for the detection of vimentin; and b) asecond probe for the detection of a multidrug resistance marker selectedfrom the group consisting of nucleophosmin and HSC70.
 21. A kit fordiagnosing or detecting multidrug resistance in a test neoplastic cellcomprising: a) a first probe for the detection of vimentin; and b) asecond probe for the detection of a marker selected from the groupconsisting of MDR1, MDR3, MRP1, MRP5, and LRP.
 22. The kit of claim 21,wherein the probe for detecting vimentin is an anti-vimentin antibody.23. The kit of claim 21, wherein the probe for detecting vimentin is avimentin ligand selected from the group consisting of LDL, NLK1 protein,vimentin, desmin, glial fibrillary acidic protein, and peripherin,fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A.
 24. Thekit of claim 20, wherein the second probe is selected from the groupconsisting of a nucleophosmin antibody and an HSC70 antibody.
 25. Thekit of claim 20, wherein the second probe is selected from the groupconsisting of a nucleophosmin ligand and an HSC70 ligand.
 26. The kit ofclaim 21, wherein the first probe detects vimentin present on thesurface of the test neoplastic cell.
 27. The kit of claim 21, whereinthe second probe detects a marker present of the surface of the testneoplastic cell.
 28. The kit of claim 21, wherein the second probe isselected from the group consisting of: an MDR1 antibody, an MDR3antibody, an MRP1 antibody, an MRP3 antibody, and an LRP antibody.
 29. Acell surface vimentin in situ detection probe for the detection of cellsurface vimentin in a patient, comprising a vimentin binding componentand a detectable label for detection in situ.
 30. The cell surfacevimentin in situ detection probe of claim 29, wherein the vimentinbinding component is an antibody.
 31. The cell surface vimentin in situdetection probe of claim 29, wherein the detectable label is Technetium.32. A cell surface vimentin-targeted agent for treating or preventing amulti-drug resistant neoplasm, comprising a vimentin binding componentand a therapeutic component, wherein the vimentin binding componenttargets the therapeutic component to the multi-drug resistant neoplasmand thereby treats the multi-drug resistant neoplasm.
 33. The agent ofclaim 32, wherein the vimentin binding component is an anti-vimentinantibody.
 34. The agent of claim 32, wherein the vimentin bindingcomponent is selected from the group consisting of LDL, NLK1 protein,vimentin, desmin, glial fibrillary acidic protein, peripherin, fimbrin,RhoA-binding kinase alpha, and protein phosphatase 2A.
 35. The agent ofclaim 32, wherein said vimentin binding component is selected from thegroup consisting of natural ligands, synthetic small molecules,chemicals, nucleic acids, peptides, proteins, antibodies, and vimentinbinding fragments thereof.
 36. The agent of claim 32, wherein thetherapeutic component is selected from the group consisting ofActinomycin, Adriamycin, Altretamine, Asparaginase, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin, Epoetin, Etoposide, Fludarabine,Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide,Imatinib, Irinotecan, Lomustine, Mechlorethamine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone,Paclitaxel, Pentostatin, Procarbazine, Taxol, Teniposide, Topotecan,Vinblastine, Vincristine, and Vinorelbine.
 37. The agent of claim 32,wherein the therapeutic component is in a liposome formulation.
 38. Theagent of claim 32, wherein the therapeutic component is a radioisotope.39. The agent of claim 38, wherein the radioisotope is selected from thegroup consisting of ⁹⁰Y, ¹²⁵I, ¹³¹I, ²¹¹At, and ²¹³Bi.
 40. The agent ofclaim 32, wherein the therapeutic component is a toxin capable ofkilling or inducing the killing of the targeted multi-drug resistantneoplastic cell.
 41. The agent of claim 40, wherein the toxin isselected from the group consisting of a Pseudomonas exotoxin, adiphtheria toxin, a plant ricin toxin, a plant abrin toxin, a plantsaporin toxin, a plant gelonin toxin, and pokeweed antiviral protein.42. The agent of claim 32, wherein the vimentin binding component bindsto the surface of the target cell and the therapeutic element isinternalized and arrests growth of the cell, compromises viability ofthe cell or kills the cell.
 43. A vaccine for treating or preventing amulti-drug resistant neoplasm, comprising a vimentin polypeptide, orvimentin polypeptide subsequence thereof, and at least onepharmaceutically acceptable vaccine component.
 44. The vaccine of claim43, wherein the vimentin polypeptide or polypeptide subsequence is ahuman vimentin polypeptide sequence of SEQ ID NO.:
 1. 45. The vaccine ofclaim 43, wherein the vimentin polypeptide subsequence is at least eightamino acids long.
 46. The vaccine of claim 45, wherein the vimentinpolypeptide subsequence comprises a hapten.
 47. The vaccine of claim 43,wherein the pharmaceutically acceptable vaccine component is anadjuvant.
 48. The vaccine of claim 47, wherein the adjuvant is selectedfrom the group consisting of aluminum hydroxide, aluminum phosphate,calcium phosphate, oil emulsion, a bacterial product, whole inactivatedbacteria, an endotoxins, cholesterol, a fatty acid, an aliphatic amine,a paraffinic compound, a vegetable oil, monophosphoryl lipid A, asaponin, and squalene.
 49. A method of treating or preventing amultidrug resistant neoplasm in a subject comprising administering acell surface vimentin-targeted therapeutic agent of claim
 32. 50. Themethod of claim 49, wherein the neoplasm is selected from the groupconsisting of a breast cancer, an ovarian cancer, a myeloma, a lymphoma,a melanoma, a sarcoma, a leukemia, a retinoblastoma, a hepatoma, aglioma, a mesothelioma, and a carcinoma.
 51. The method of claim 49,wherein the subject is a human patient.
 52. The method of claim 51,wherein the human patient is suffering from a disease or disorder causedby the presence of the multi-drug resistant cell.
 53. The method ofclaim 49, wherein the neoplasm is from a tissue selected from the groupconsisting of blood, bone marrow, spleen, lymph node, liver, thymus,kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, andovary.
 54. A method of treating or preventing a multidrug resistantneoplasm in a subject comprising administering a vimentin vaccine ofclaim
 43. 55. The method of claim 54, wherein the neoplasm is selectedfrom the group consisting of a breast cancer, an ovarian cancer, amyeloma, a lymphoma, a melanoma, a sarcoma, a leukemia, aretinoblastoma, a hepatoma, a glioma, a mesothelioma, and a carcinoma.56. The method of claim 54, wherein the subject is a human patient. 57.The method of claim 56, wherein the human patient is suffering from adisease or disorder caused by the presence of the multi-drug resistantcell.
 58. The method of claim 54, wherein the neoplasm is from a tissueselected from the group consisting of blood, bone marrow, spleen, lymphnode, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye,breast, prostate, and ovary. 59-65. (canceled)
 66. A method fordetecting a neoplastic cell in a patient comprising: (a) administeringto the patient, a vimentin binding agent operably linked to a detectablelabel; and (b) detecting the label operably linked to the vimentinbinding agent, wherein the vimentin binding agent specifically binds tocell surface-expressed vimentin present on a neoplastic cell in thepatient.
 67. The method of claim 66, wherein the vimentin binding agentis an antibody or fragment thereof.
 68. The method of claim 66, whereinthe vimentin binding agent is selected from the group consisting ofmodified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidicprotein, and peripherin, fimbrin, RhoA-binding kinase alpha, and proteinphosphatase 2A.
 69. The method of claim 66, wherein the vimentin bindingagent is selected from the group consisting of natural ligands,synthetic small molecules, chemicals, nucleic acids, peptides, proteins,antibodies, and fragments thereof.
 70. The method of claim 66, whereinthe detectable label is selected from the group consisting offluorophores, chemical dyes, radioactive compounds, chemoluminescentcompounds, magnetic compounds, paramagnetic compounds, promagneticcompounds, enzymes that yield a colored product, enzymes that yield achemoluminescent product, and enzymes that yield a magnetic product. 71.The method of claim 66, wherein the neoplastic cell is selected from thegroup consisting of a breast cancer cell, an ovarian cancer cell, amyeloma cancer cell, a lymphoma cancer cell, a melanoma cancer cell, asarcoma cancer cell, a leukemia cancer cell, a retinoblastoma cancercell, a hepatoma cancer cell, a glioma cancer cell, a mesotheliomacancer cell, and a carcinoma cancer cell.
 72. The method of claim 66,wherein the neoplastic cell is selected from the group consisting of apromyleocytic leukemia cell, a T lymphoblastoid cell, a breastepithelial cell, and an ovarian cell.
 73. The method of claim 66,wherein the patient is a human.
 74. The method of claim 73, wherein thepatient is suffering from a disease or disorder caused by the presenceof the neoplastic cell.
 75. A kit for diagnosing or detecting neoplasia,comprising: a) a first probe for the detection of vimentin; and b) asecond probe for the detection of a neoplasia marker selected from thegroup consisting of nucleophosmin and HSC70.
 76. The kit of claim 75,wherein the probe for detecting vimentin is an anti-vimentin antibody orbinding fragment thereof.
 77. The kit of claim 75, wherein the probe fordetecting vimentin is a vimentin ligand selected from the groupconsisting of LDL, NLK1 protein, vimentin, desmin, glial fibrillaryacidic protein, and peripherin, fimbrin, RhoA-binding kinase alpha, andprotein phosphatase 2A.
 78. The kit of claim 75, wherein the secondprobe is selected from the group consisting of a nucleophosmin antibodyand an HSC70 antibody.
 79. The kit of claim 75, wherein the second probeis selected from the group consisting of a nucleophosmin ligand and anHSC70 ligand.
 80. The kit of claim 75, wherein the first probe detectsvimentin present on the surface of the test cell if it is neoplastic.81. The kit of claim 75, wherein the second probe detects a markerpresent of the surface of the test cell if it is neoplastic.
 82. A cellsurface vimentin-targeted agent for treating a cancerous neoplastic cellgrowth comprising a vimentin binding component and a therapeuticcomponent, wherein the vimentin binding component targets thetherapeutic component to the neoplastic cell growth and thereby treatsthe cancer.
 83. The agent of claim 82, wherein the vimentin bindingcomponent is an anti-vimentin antibody.
 84. The agent of claim 82,wherein the vimentin binding component is selected from the groupconsisting of LDL, NLK1 protein, vimentin, desmin, glial fibrillaryacidic protein, and peripherin, fimbrin, RhoA-binding kinase alpha, andprotein phosphatase 2A.
 85. The agent of claim 82, wherein said vimentinbinding component is selected from the group consisting of naturalligands, synthetic small molecules, chemicals, nucleic acids, peptides,proteins, antibodies, and vimentin binding fragments thereof.
 86. Theagent of claim 82, wherein the therapeutic component is selected fromthe group consisting of Actinomycin, Adriamycin, Altretamine,Asparaginase, Bleomycin, Busulfan, Capecitabine, Carboplatin,Carmustine, Chlorambucil, Cisplatin, Cladribine, Cyclophosphamide,Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Docetaxel,Doxorubicin, Epoetin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Ifosfamide, Imatinib, Irinotecan, Lomustine,Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin,Mitotane, Mitoxantrone, Paclitaxel, Pentostatin, Procarbazine, Taxol,Teniposide, Topotecan, Vinblastine, Vincristine, and Vinorelbine andcombinations thereof.
 87. The agent of claim 82, wherein the therapeuticcomponent is in a liposome formulation.
 88. The agent of claim 82,wherein the therapeutic component is a radioisotope.
 89. The agent ofclaim 88, wherein the radioisotope is selected from the group consistingof ⁹⁰Y, ¹¹¹In, ¹²⁵I, ¹³¹I, ²¹¹At, and ²¹³Bi.
 90. The agent of claim 82,wherein the therapeutic component is a toxin capable of killing orinducing the killing of the targeted neoplastic cell.
 91. The agent ofclaim 90, wherein the toxin is selected from the group consisting of aPseudomonas exotoxin, a diphtheria toxin, a plant ricin toxin, a plantabrin toxin, a plant saporin toxin, a plant gelonin toxin, and pokeweedantiviral protein.
 92. The agent of claim 82, wherein the vimentinbinding component binds to the surface of the target cell and thetherapeutic element is internalized and arrests growth of the cell,compromises viability of the cell, or kills the cell.
 93. A vaccine fortreating or preventing a neoplasm comprising a vimentin polypeptide, orvimentin polypeptide subsequence thereof, and at least onepharmaceutically acceptable vaccine component.
 94. The vaccine of claim93, wherein the vimentin polypeptide or polypeptide subsequence is ahuman vimentin polypeptide sequence set forth in SEQ ID NO.:
 1. 95. Thevaccine of claim 93, wherein the vimentin polypeptide subsequence is atleast eight amino acids long.
 96. The vaccine of claim 95, wherein thevimentin polypeptide subsequence comprises a hapten.
 97. The vaccine ofclaim 93, wherein the pharmaceutically acceptable vaccine component isan adjuvant.
 98. The vaccine of claim 97, wherein the adjuvant isselected from the group consisting of aluminum hydroxide, aluminumphosphate, calcium phosphate, oil emulsion, a bacterial product, wholeinactivated bacteria, an endotoxins, cholesterol, a fatty acid, analiphatic amine, a paraffinic compound, a vegetable oil, monophosphoryllipid A, a saponin, and squalene.
 99. A method of treating or preventinga neoplasm in a subject comprising administering a cell surfacevimentin-targeted therapeutic agent of claim
 82. 100. The method ofclaim 99, wherein the neoplasm is selected from the group consisting ofa breast cancer, an ovarian cancer, a myeloma, a lymphoma, a melanoma, asarcoma, a leukemia, a retinoblastoma, a hepatoma, a glioma, amesothelioma, and a carcinoma.
 101. The method of claim 99, wherein thesubject is a human patient.
 102. The method of claim 101, wherein saidhuman patient is suffering from a disease or disorder caused by thepresence of the multi-drug resistant cell.
 103. The method of claim 99,wherein the neoplasm is from a tissue selected from the group consistingof blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain,skin, gastrointestinal tract, eye, breast, prostate and ovary.
 104. Amethod of treating or preventing a neoplasm in a subject comprisingadministering a vimentin vaccine of claim
 93. 105. The method of claim104, wherein the neoplasm is selected from the group consisting of abreast cancer, an ovarian cancer, a myeloma, a lymphoma, a melanoma, asarcoma, a leukemia, a retinoblastoma, a hepatoma, a glioma, amesothelioma, and a carcinoma.
 106. The method of claim 104, whereinsaid subject is a human patient.
 107. The method of claim 106, whereinsaid human patient is suffering from a disease or disorder caused by thepresence of the neoplastic cell.
 108. The method of claim 104, whereinthe neoplasm is from a tissue selected from the group consisting ofblood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain,skin, gastrointestinal tract, eye, breast, prostate, and ovary.
 109. Themethod of claim 10, wherein the vimentin binding agent is ananti-vimentin antibody.
 110. The method of claim 109, wherein theanti-vimentin antibody is operably linked to a radiolabel.